Control Of Amino Acid Synthesis Research Articles

Mitochondrial CypD Acetylation Promotes Endothelial Dysfunction and Hypertension.

Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial CypD (cyclophilin D) at K166 contributes to endothelial dysfunction and hypertension. To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial-specific GCN5L1 (general control of amino acid synthesis 5 like 1)-deficient mice using an Ang II (angiotensin II) model of hypertension. Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 (sirtuin 3) and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation, which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells, GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension. Ang II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in a 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in the endothelial function was tested in endothelial-specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented Ang II-induced mitochondrial oxidative stress, reduced the maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation, which may be beneficial in cardiovascular disease.

Loss of GCN5L1 exacerbates damage in alcoholic liver disease through ferroptosis activation.

Iron overload, oxidative stress and ferroptosis are associated with liver injury in alcohol-associated liver disease (ALD), however, the crosstalk among these regulatory pathways in ALD development is unclear. ALD mouse model and general control of amino acid synthesis 5 like 1 (GCN5L1) liver knockout mice were generated to investigate the role of GCN5L1 in ALD development. Proteomic screening tests were performed to identify the key factors mediating GCN5L1 loss-induced ALD. Gene Expression Omnibus data set analysis indicates that GCN5L1 expression is negatively associated with ALD progression. GCN5L1 hepatic knockout mice develop severe liver injury and lipid accumulation when fed an alcohol diet. Screening tests identified that GCN5L1 targeted the mitochondrial iron transporter CISD1 to regulate mitochondrial iron homeostasis in ethanol-induced ferroptosis. GCN5L1-modulated CISD1 acetylation and activity were crucial for iron accumulation and ferroptosis in response to alcohol exposure. Pharmaceutical modulation of CISD1 activity is critical for cellular iron homeostasis and ethanol-induced ferroptosis. The GCN5L1/CISD1 axis is crucial for oxidative stress and ethanol-induced ferroptosis in ALD and is a promising avenue for novel therapeutic strategies.

GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells

BackgroundThe pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation.MethodGCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling.ResultsKnocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations.ConclusionsIn summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Retrograde regulation of mitochondrial fission and epithelial to mesenchymal transition in hepatocellular carcinoma by GCN5L1.

Metabolic reprogram is crucial to support cancer cell growth and movement as well as determine cell fate. Mitochondrial protein acetylation regulates mitochondrial metabolism, which is relevant to cancer cell migration and invasion. The functional role of mitochondrial protein acetylation on cancer cell migration remains unclear. General control of amino acid synthesis 5 like-1(GCN5L1), as the regulator of mitochondrial protein acetylation, functions on metabolic reprogramming in mouse livers. In this study, we find that GCN5L1 expression is significantly decreased in metastatic HCC tissues. Loss of GCN5L1 promotes reactive oxygen species (ROS) generation through enhanced fatty acid oxidation (FAO), followed by activation of cellular ERK and DRP1 to promote mitochondrial fission and epithelia to mesenchymal transition (EMT) to boost cell migration. Moreover, palmitate and carnitine-stimulated FAO promotes mitochondrial fission and EMT gene expression to activate HCC cell migration. On the other hand, increased cellular acetyl-CoA level, the product of FAO, enhances HCC cell migration. Taken together, our finding uncovers the metastasis suppressor role as well as the underlying mechanism of GCN5L1 in HCC and also provides evidence of FAO retrograde control of HCC metastasis.

Abstract 13610: Imbalance Between GCN5L1 Mediated CypD Acetylation and Sirt3 Deacetylase Promotes Mitochondrial Oxidative Stress, Endothelial Dysfunction and Hypertension

Nearly half of adult population has hypertension which is a main risk factor for cardiovascular disease. Mitochondrial dysfunction contributes to hypertension and targeting mitochondria can potentially improve treatment of hypertension. We found hyperacetylation of mitochondrial Cyclophilin D (CypD) in essential hypertension and proposed that CypD acetylation can promote endothelial dysfunction and hypertension. CypD acetylation is mediated by general control of amino acid synthesis 5 like 1 (GCN5L1) protein which is counteracted by deacetylase Sirt3. We tested potential role GCN5L1 and Sirt3 in endothelial dysfunction and hypertension. Western blot of aortic mitochondria showed an increased GCN5L1 level in hypertensive mice coupled with the reduction of Sirt3 deacetylase resulting in 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. We reported pathogenic role of lipid oxidation products isolevuglandins (isoLGs) and scavenging isoLGs by mitochondria-targeted mito2HOBA reduces mitochondrial acetylation. We tested if mitochondrial isoLGs contributes to acetylase/deacetylase imbalance driving CypD acetylation. Treatment with mito2HOBA improves Sirt3 expression and reduces GCN5L1 level resulting in normalization of GCN5L1/Sirt3 ratio and reduced CypD acetylation. To define the pathophysiological role of GCN5L1 in endothelial cells we tested if GCN5L1 depletion prevents cytokine-induced of mitochondrial oxidative stress. Indeed, treatment of human aortic endothelial cells with AngII+TNFα induces mitochondrial O 2 . -, however, GCN5L1 depletion using siRNA completely abrogated the cytokine-induced mitochondrial oxidative stress. To define the role GNC5L1 in pathogenesis of endothelial dysfunction we developed new tamoxifen-inducible endothelial specific GNC5L1 knockout mice (Ec GNC5L1KO ). Depletion of endothelial GCN5L1 reduces vascular mitochondrial O 2 . -, prevents inactivation of endothelial nitric oxide and preserves endothelial dependent relaxation in angiotensin II-infused Ec GNC5L1KO mice compared with wild-type littermates. These data support the pathogenic role of GNC5L1 in CypD acetylation and endothelial dysfunction and targeting GNC5L1 can be beneficial in cardiovascular disease.

Abstract P237: Mitochondrial GCN5L1 Promotes CypD Acetylation, Oxidative Stress, Endothelial Dysfunction And Hypertension

Nearly half of adult population has hypertension which is a main risk factor for cardiovascular disease. Mitochondrial dysfunction contributes to hypertension and targeting mitochondria can potentially improve treatment of hypertension. We found hyperacetylation of mitochondrial Cyclophilin D (CypD) in essential hypertension and proposed that CypD acetylation can promote endothelial dysfunction and hypertension. CypD acetylation can be regulated by general control of amino acid synthesis 5 like 1 (GCN5L1) protein, therefore, we GCN5L1 can potentially promote endothelial dysfunction and hypertension. Western blot of aortic mitochondria showed an increased GCN5L1 level in hypertensive mice which was coupled with the reduction of Sirt3 deacetylase level resulting in 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. We reported pathogenic role of mitochondrial lipid oxidation products isolevuglandins (isoLGs) and scavenging of isoLGs with mitochondria-targeted mito2HOBA reduces mitochondrial acetylation. We tested if mitochondrial isoLGs contributes to acetylase/deacetylase imbalance driving CypD acetylation. It was found that mito2HOBA supplementation improves Sirt3 expression and reduces GCN5L1 level leading to normalization of GCN5L1/Sirt3 ratio and reduced CypD acetylation. To define the pathophysiological role of GCN5L1 in endothelial cells we tested if GCN5L1 depletion prevents cytokine-induced of mitochondrial oxidative stress. Indeed, treatment of human aortic endothelial cells with AngII+TNFα induces mitochondrial O 2 . -, however, GCN5L1 depletion using siRNA completely abrogated the cytokine-induced mitochondrial oxidative stress. To define the role GNC5L1 in pathogenesis of endothelial dysfunction we have developed new tamoxifen-inducible endothelial specific GNC5L1 knockout mice (Ec GNC5L1KO ). It was found that depletion of endothelial GCN5L1 reduces vascular production of mitochondrial O 2 . -, prevents inactivation of endothelial nitric oxide and preserves endothelial dependent relaxation in angiotensin II-infused Ec GNC5L1KO mice compared with wild-type littermates. These data support the pathogenic role of GNC5L1 in CypD acetylation and development of endothelial dysfunction.

Mitochondrial GCN5L1 regulates cytosolic redox state and hepatic gluconeogenesis via glycerol phosphate shuttle GPD2

Hepatic gluconeogenesis is crucial for maintaining blood glucose during starvation, and a major contributor for hyperglycemia. Cellular redox state is related to mitochondrial biology and regulates conversion of specific metabolites to glucose. General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) is a mitochondria-enriched protein which modulates glucose and amino acid metabolism. Here we show a new regulatory mode of GCN5L1 on gluconeogenesis using lactate and glycerol. We observed GCN5L1 deletion dramatically inhibited glucose production derived from glycerol and lactate, due to increased cytosolic redox state. The underlying mechanism is that GCN5L1 directly binds to the key component of mitochondrial shuttle glycerol phosphate dehydrogenase 2 (GPD2) and modulates its activity. These results have significant implications for understanding the physiological role and regulatory mechanism of mitochondrial shuttle in diabetes development and provide a novel therapeutic potential for diabetes.

Mitochondrial GCN5L1 regulates glutaminase acetylation and hepatocellular carcinoma.

BackgroundGlutaminolysis is a critical metabolic process that promotes cancer cell proliferation, including hepatocellular carcinoma (HCC). Delineating the molecular control of glutaminolysis could identify novel targets to ameliorate this oncogenic metabolic pathway. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a regulator of mitochondrial protein acetylation, in modulating the acetylation and activity of glutaminase to regulate HCC development.MethodsCell proliferation was determined by MTT, 2D and soft agar clone formation assays and orthotopic tumour assays in nude mice. GLS1/2 acetylation and activities were measured in cells and tumours to analyse the correlation with GCN5L1 expression and mTORC1 activation.ResultsHepatic GCN5L1 ablation in mice markedly increased diethylnitrosamine (DEN)‐induced HCC, and conversely, the transduction of mitochondrial‐restricted GCN5L1 protected wild‐type mice against HCC progression in response to DEN and carbon tetrachloride (CCl4) exposure. GCN5L1–depleted HepG2 hepatocytes enhanced tumour growth in athymic nude mice. Mechanistically, GCN5L1 depletion promoted cell proliferation through mTORC1 activation. Interestingly, liver–enriched glutaminase 2 (GLS2) appears to play a greater role than ubiquitous and canonical tumour–enriched glutaminase 1 (GLS1) in promoting murine HCC. Concurrently, GCN5L1 promotes acetylation and inactivation of both isoforms and increases enzyme oligomerisation. In human HCC tumours compared to adjacent tissue, there were variable levels of mTORC1 activation, GCN5L1 levels and glutaminase activity. Interestingly, the levels of GCN5L1 inversely correlated with mTORC1 activity and glutaminase activity in these tumours.ConclusionsOur study identified that glutaminase activity, rather than GLS1 or GLS2 expression, is the key factor in HCC development that activates mTORC1 and promotes HCC. In the Kaplan–Meier analysis of liver cancer, we found that HCC patients with high GCN5L1 expression survived longer than those with low GCN5L1 expression. Collectively, GCN5L1 functions as a tumour regulator by modulating glutaminase acetylation and activity in the development of HCC.

Centromere Protein A (CENPA) Regulates Metabolic Reprogramming in the Colon Cancer Cells by Transcriptionally Activating Karyopherin Subunit Alpha 2 (KPNA2)

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

Combined overexpression of SIRT1 and knockout of GCN5 in adult skeletal muscle does not affect glucose homeostasis or exercise performance in mice.

Sirtuin 1 (SIRT1) and general control of amino acid synthesis 5 (GCN5) regulate mitochondrial biogenesis via opposing modulation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) acetylation status and activity. However, the combined contribution of SIRT1 and GCN5 to skeletal muscle metabolism and endurance performance in vivo is unknown. In this study, we investigated the impact of combined skeletal muscle-specific overexpression of SIRT1 and deletion of GCN5 on glucose homeostasis, skeletal muscle mitochondrial biogenesis and function, and metabolic adaptation to endurance exercise training in mice. We generated mice with combined and tamoxifen-inducible skeletal muscle-specific overexpression of SIRT1 and knockout of GCN5 (dTG) and floxed [wild type (WT)] littermates using a Cre-LoxP approach. All mice were treated with tamoxifen at 5-6 wk of age, and 4-7 wk later glucose homeostasis, skeletal muscle contractile function, mitochondrial function, and the effects of 14 days of voluntary wheel running on expression of metabolic proteins and exercise capacity were assessed. There was no difference in oral glucose tolerance, skeletal muscle contractile function, mitochondrial abundance, or maximal respiratory capacity between dTG and WT mice. Additionally, there were no genotype differences in exercise performance and markers of mitochondrial biogenesis after 14 days of voluntary wheel running. These results demonstrate that combined overexpression of SIRT1 and loss of GCN5 in vivo does not promote metabolic remodeling in skeletal muscle of sedentary or exercise-trained mice.

Mitochondrial General Control of Amino Acid Synthesis 5 Like 1 Regulates Glutaminolysis, Mammalian Target of Rapamycin Complex 1 Activity, and Murine Liver Regeneration

The regenerative capacity of the liver plays a protective role against hepatotoxins and impaired regeneration exacerbates liver dysfunction in nonalcoholic fatty liver disease (NAFLD). Mitochondrial bioenergetic and -synthetic functions are important contributory factors in hepatic regeneration, and the control of mitochondrial protein acetylation is implicated in the mitochondrial susceptibility to liver stressors. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a mediator of mitochondrial metabolism and acetylation, in modulating murine liver regeneration (LR) in response to acute CCl4 -induced hepatotoxicity. Initial metabolomic screening found that liver GCN5L1 knockout (LKO) mice have augmented glutaminolysis. Absence of GCN5L1 modified enzyme activity of liver-enriched glutaminase enzyme (glutaminase 2; GLS2), and GCN5L1 levels modulated GLS2 oligomerization and acetylation. This metabolic remodeling resulted in the elevation of α-ketoglutarate levels, which are known to activate mammalian target of rapamycin complex 1 (mTORC1). This signaling pathway was induced with increased phosphorylation of S6 kinase in LKO hepatocytes, and inhibition of glutaminolysis reversed aberrant mTORC1 signaling. At the same time, glutaminolysis, activity of GLS2, and activation of mTORC1 signaling were reversed by the genetic reintroduction of the mitochondrial isoform of GCN5L1 into LKO primary hepatocytes. Finally, LKO mice had a more robust regenerative capacity in response to CCl4 hepatoxicity, and this response was blunted by both the mTORC1 inhibitor, rapamycin, and by pharmacological blunting of glutaminolysis. These data point to a central role of glutaminolysis in modulating the regenerative capacity in the liver. Furthermore, inhibition of mitochondrial GCN5L1 to augment LR may be a useful strategy in disease states linked to hepatotoxicity.

Molecular Regulations of Mucosal Maltase Expressions.

Two major α-glucosidase (maltase) genes, sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM), respectively, are expressed in the small intestine. In this review, we have summarized whether jejunal expression of these maltase genes is regulated by dietary manipulations, which may affect carbohydrate availability from the luminal side, through changes in the binding of transcription factors and/or histone code on these genes. Studies using a model of mice fed either a low-starch or a high-starch diet for 7 days, found the mRNA levels of SI, MGAM, and Na-glucose cotransporter (SGLT1) genes in the jejunum to be increased in parallel by feeding a high-starch diet. Chromatin immunoprecipitation assays, using jejunal tissue of mice and rats fed a high-starch diet, revealed that the diet increased the acetylations of histones H3 and H4, bindings of coactivators, including general control of amino acid synthesis (GCN5) and the transcriptional factors, including caudal-related homeobox 2 (CDX2), and hepatocyte nuclear factor 1 (HNF1), not only in the promoter/enhancer regions, but also in the transcribed regions of SI and MGAM genes. Feeding rats a diet rich in resistant starch led to a concomitant reduction of mRNA levels of the MGAM gene and histone H3 modifications (acetylations and di-/tri-methylations) in the jejunum. These data suggest that a signal elicited by available glucose in the jejunal mucosa is associated with SI and MGAM gene expressions through a histone code, such as acetylation and di-/tri-methylations of histone H3 in the promoter/enhancer and transcribed regions of SI and MGAM genes.

GCN5L1/BLOS1 Links Acetylation, Organelle Remodeling, and Metabolism.

General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) was identified as a novel gene with sequence homology to the histone acetyltransferase Gcn5. Subsequent protein-interaction studies identified GCN5L1 as a subunit of the multiprotein lysosome biogenesis complex, resulting in an alternative designation as biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1 or BLOC1S1). Despite the distinct nomenclatures, GCN5L1/BLOS1 has been shown to play crucial roles in mitochondria, endosomes, lysosomes, and synaptic vesicle precursors (SVPs). GCN5L1/BLOS1 controls mitochondrial protein acetylation, modulates metabolic pathways, and orchestrates retrograde mitochondria-to-nucleus signaling. It also contributes to endosome-lysosome and vesicle trafficking and to endolysosomal function. Here we discuss the intracellular roles of GCN5L1/BLOS1 in the hope of linking mitochondria-centric effects to cytosolic vesicle biology.

Muscle-specific knockout of general control of amino acid synthesis 5 (GCN5) does not enhance basal or endurance exercise-induced mitochondrial adaptation

ObjectiveLysine acetylation is an important post-translational modification that regulates metabolic function in skeletal muscle. The acetyltransferase, general control of amino acid synthesis 5 (GCN5), has been proposed as a regulator of mitochondrial biogenesis via its inhibitory action on peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α). However, the specific contribution of GCN5 to skeletal muscle metabolism and mitochondrial adaptations to endurance exercise in vivo remain to be defined. We aimed to determine whether loss of GCN5 in skeletal muscle enhances mitochondrial density and function, and the adaptive response to endurance exercise training. MethodsWe used Cre-LoxP methodology to generate mice with muscle-specific knockout of GCN5 (mKO) and floxed, wildtype (WT) littermates. We measured whole-body energy expenditure, as well as markers of mitochondrial density, biogenesis, and function in skeletal muscle from sedentary mice, and mice that performed 20 days of voluntary endurance exercise training. ResultsDespite successful knockdown of GCN5 activity in skeletal muscle of mKO mice, whole-body energy expenditure as well as skeletal muscle mitochondrial abundance and maximal respiratory capacity were comparable between mKO and WT mice. Further, there were no genotype differences in endurance exercise-mediated mitochondrial biogenesis or increases in PGC-1α protein content. ConclusionThese results demonstrate that loss of GCN5 in vivo does not promote metabolic remodeling in mouse skeletal muscle.

Acetylation and succinylation contribute to maturational alterations in energy metabolism in the newborn heart.

Dramatic maturational changes in cardiac energy metabolism occur in the newborn period, with a shift from glycolysis to fatty acid oxidation. Acetylation and succinylation of lysyl residues are novel posttranslational modifications involved in the control of cardiac energy metabolism. We investigated the impact of changes in protein acetylation/succinylation on the maturational changes in energy metabolism of 1-, 7-, and 21-day-old rabbit hearts. Cardiac fatty acid β-oxidation rates increased in 21-day vs. 1- and 7-day-old hearts, whereas glycolysis and glucose oxidation rates decreased in 21-day-old hearts. The fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD) and β-hydroxyacyl-CoA dehydrogenase (β-HAD), were hyperacetylated with maturation, positively correlated with their activities and fatty acid β-oxidation rates. This alteration was associated with increased expression of the mitochondrial acetyltransferase, general control of amino acid synthesis 5 like 1 (GCN5L1), since silencing GCN5L1 mRNA in H9c2 cells significantly reduced acetylation and activity of LCAD and β-HAD. An increase in mitochondrial ATP production rates with maturation was associated with the decreased acetylation of peroxisome proliferator-activated receptor-γ coactivator-1α, a transcriptional regulator for mitochondrial biogenesis. In addition, hypoxia-inducible factor-1α, hexokinase, and phosphoglycerate mutase expression declined postbirth, whereas acetylation of these glycolytic enzymes increased. Phosphorylation rather than acetylation of pyruvate dehydrogenase (PDH) increased in 21-day-old hearts, accounting for the low glucose oxidation postbirth. A maturational increase was also observed in succinylation of PDH and LCAD. Collectively, our data are the first suggesting that acetylation and succinylation of the key metabolic enzymes in newborn hearts play a crucial role in cardiac energy metabolism with maturation.

Observing Changes in the Structure and Oligomerization State of a Helical Protein Dimer Using Solid-State Nanopores.

Protein analysis using solid-state nanopores is challenging due to limitations in bandwidth and signal-to-noise ratio. Recent improvements of those two aspects have made feasible the study of small peptides using solid-state nanopores, which have an advantage over biological counterparts in tunability of the pore diameter. Here, we report on the detection and characterization of peptides as small as 33 amino acids. Silicon nitride nanopores with thicknesses less than 10 nm are used to provide signal-to-noise (S/N) levels up to S/N ∼ 10 at 100 kHz. We demonstrate differentiation of monomer and dimer forms of the GCN4-p1 leucine zipper, a coiled-coil structure well studied in molecular biology, and compare with the unstructured 33-residue monomer. GCN4-p1 is sequence segment associated with homodimerization of the transcription factor General Control Nonderepressible 4 (GCN4), which is involved in the control of amino acid synthesis in yeast. The differentiation between two oligomeric forms demonstrates the capabilities of improved solid-state nanopore platforms to extract structural information involving short peptide structures.

Abstract 18405: Epidrug Treatment Rescues Proliferation and Differentiation in Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients: A Case of Epimetabolic Memory

Introduction: The origin of metabolic/epigenetic memory remains elusive. It, however, may be at the basis of chronic diseases and functional alteration associated to ageing and cardiovascular disease. HYPOTHESIS: Diabetes is known to alter cellular metabolism causing defects in tissue/organ regeneration. In this study we asked whether human cardiac stromal cells from diabetic and normoglycaemic voluntary donors revealed differences associated to their original metabolism and whether the alterations could be rescued by epigenetically active drugs. METHODS and RESULTS: D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of histone H3 lysine 9 and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5- related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-a general control of amino acid synthesis (GCN5a), determining a relative decreasein total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. CONCLUSIONS: This study investigated the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Aberrant levels of histone H3 acetylation induce spermatid anomaly in mouse testis

Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.

Obesity-induced lysine acetylation increases cardiac fatty acid oxidation and impairs insulin signalling.

Lysine acetylation is a novel post-translational pathway that regulates the activities of enzymes involved in both fatty acid and glucose metabolism. We examined whether lysine acetylation controls heart glucose and fatty acid oxidation in high-fat diet (HFD) obese and SIRT3 knockout (KO) mice. C57BL/6 mice were placed on either a HFD (60% fat) or a low-fat diet (LFD; 4% fat) for 16 or 18 weeks. Cardiac fatty acid oxidation rates were significantly increased in HFD vs. LFD mice (845 ± 76 vs. 551 ± 87 nmol/g dry wt min, P < 0.05). Activities of the fatty acid oxidation enzymes, long-chain acyl-CoA dehydrogenase (LCAD), and β-hydroxyacyl-CoA dehydrogenase (β-HAD) were increased in hearts from HFD vs. LFD mice, and were associated with LCAD and β-HAD hyperacetylation. Cardiac protein hyperacetylation in HFD-fed mice was associated with a decrease in SIRT3 expression, while expression of the mitochondrial acetylase, general control of amino acid synthesis 5 (GCN5)-like 1 (GCN5L1), did not change. Interestingly, SIRT3 deletion in mice also led to an increase in cardiac fatty acid oxidation compared with wild-type (WT) mice (422 ± 29 vs. 291 ± 17 nmol/g dry wt min, P < 0.05). Cardiac lysine acetylation was increased in SIRT3 KO mice compared with WT mice, including increased acetylation and activity of LCAD and β-HAD. Although the HFD and SIRT3 deletion decreased glucose oxidation, pyruvate dehydrogenase acetylation was unaltered. However, the HFD did increase Akt acetylation, while decreasing its phosphorylation and activity. We conclude that increased cardiac fatty acid oxidation in response to high-fat feeding is controlled, in part, via the down-regulation of SIRT3 and concomitant increased acetylation of mitochondrial β-oxidation enzymes.

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.