Increase In Protein Acetylation Research Articles

Age-related disruption of the proteome and acetylome in mouse hearts is associated with loss of function and attenuated by elamipretide (SS-31) and nicotinamide mononucleotide (NMN) treatment.

We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes associated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results identify a subset of protein abundance and acetylation changes in muscle, mitochondrial, and structural proteins that appear to be essential in regulating diastolic function in old hearts.

Extended Ketogenic Diet Is Necessary For Increases In Protein Acetylation

Decline in mitochondrial function is associated with a decrease in lifespan. We have previously demonstrated that a long-term ketogenic diet (KD) improves mitochondrial function and longevity. However, a life-long KD is difficult to maintain and an intermittent KD might be more viable long term. PURPOSE: Determine how long it takes before a ketogenic diet alters muscle metabolism so that intermittent diets can be developed. METHODS: Four C57BL/6 mice were fed a control diet or 1 or 7 days of continuous KD. At the time of sacrifice, livers, gastrocnemius, brain and kidneys were extracted and frozen in liquid nitrogen before being powdered and homogenized in sucrose lysis buffer and prepared for western blot analysis to determine total acetylated lysine content, total OXPHOS protein, or acetylated p300 content. RESULTS: Following one day of KD, neither acetylated, nor mitochondrial proteins were different than control diet. By seven days of continuous KD diet, total acetylated proteins increased in the liver, kidney and gastrocnemius muscle. Specifically, acetylation of p300 was 3.4±0.89-fold greater following 7 days of KD. Unlike the other tissues the brain showed no difference in acetylated proteins by 7 days. An increase in mitochondrial mass was only seen in the liver at 7 days of KD. CONCLUSIONS: A short term ketogenic diet can be used to rapidly alter protein acetylation in the liver, kidney and muscle. These data suggest that an intermittent keto diet may be useful in promoting a biochemical change in muscle that promotes mitochondrial function and may benefit long-term muscle function.

Acetylation contributes to hypertrophy-caused maturational delay of cardiac energy metabolism.

A dramatic increase in cardiac fatty acid oxidation occurs following birth. However, cardiac hypertrophy secondary to congenital heart diseases (CHDs) delays this process, thereby decreasing cardiac energetic capacity and function. Cardiac lysine acetylation is involved in modulating fatty acid oxidation. We thus investigated what effect cardiac hypertrophy has on protein acetylation during maturation. Eighty-four right ventricular biopsies were collected from CHD patients and stratified according to age and the absence (n = 44) or presence of hypertrophy (n = 40). A maturational increase in protein acetylation was evident in nonhypertrophied hearts but not in hypertrophied hearts. The fatty acid β-oxidation enzymes, long-chain acyl CoA dehydrogenase (LCAD) and β-hydroxyacyl CoA dehydrogenase (βHAD), were hyperacetylated and their activities positively correlated with their acetylation after birth in nonhypertrophied hearts but not hypertrophied hearts. In line with this, decreased cardiac fatty acid oxidation and reduced acetylation of LCAD and βHAD occurred in newborn rabbits subjected to cardiac hypertrophy due to an aortocaval shunt. Silencing the mRNA of general control of amino acid synthesis 5-like protein 1 reduced acetylation of LCAD and βHAD as well as fatty acid oxidation rates in cardiomyocytes. Thus, hypertrophy in CHDs prevents the postnatal increase in myocardial acetylation, resulting in a delayed maturation of cardiac fatty acid oxidation.

Oroxylin A prevents cardiac hypertrophy and dysfunction as a modulator of mitochondrial protein acetylation

Cardiac hypertrophy and remodeling are both well‐known consequences mediated by increased levels of Angiotensin II (Ang II) as a compensatory mechanism in non‐ischemic HF. In this sense, mitochondrial protein hyper‐acetylation and ROS production that induce mitochondrial dysfunction have previously been associated with this condition. Oroxylin A (OA) is a methylated flavone found on the root of Scutellaria baicalensis known for its antioxidant properties, and as a modulator of mitochondrial deacetylase activity. Nevertheless, OA effects have not been explored in cardiac remodeling mediated by exacerbated ROS production and mitochondrial hyper‐acetylation. Therefore, we explore the potential cardioprotective effect of OA in an in vitro model of cardiac hypertrophy induced by Ang II treatment in H9c2 myocytes. We found that OA reverted completely the Ang II mediated hypertrophic and cell death effect. Mitochondrial acetylation profile and sirtuin3 expression analyzed by western blot showed that Ang II promotes ~2‐fold increase in protein acetylation; however, OA pre‐treatment abrogates Ang II‐induced hyper‐acetylation in a dose‐dependent fashion. Remarkably, we found that the OA pre‐treatment prevents mitochondrial dysfunction by decreasing significantly permeability transition susceptibility and membrane potential depolarization. These results were consistent with a decrease in apoptosis, determined by caspase 9 and 3,7 activity. Overall, these findings suggest that OA promotes sirtuin3 activation, targeting pivot mitochondrial proteins. Accordingly, we evaluated the cardioprotective effect of OA in an in vivo model of HF induced by AngII+L‐NAME. In this model, we found an increased mitochondrial acetylation in the myocardium and found that OA was capable of prevent cardiac hypertrophy and dysfunction. Taken together, our results suggest that OA is a novel strategy to treat cardiac dysfunction in HF.Support or Funding InformationThis work was partially supported by Xignus Research Foundation as well endowed Chair in Cardiology/Grupo de Enfoque Medicina Cardiovascular Tec de Monterrey, CONACYT Grant CB‐256577, Fronteras de la Ciencias 0682 (Gerardo García‐Rivas), and Red Temática Farmoquímicos del CONACYT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

Dietary Restriction Increases Protein Acetylation in the Livers of Aged Rats

Background: Dietary restriction (DR) is a well-established biological method for lifespan extension in various organisms by delaying the progression of age-related disorders. With regard to its molecular mechanisms, a family of NAD-dependent protein deacetylases, such as sirtuins, is considered to mediate DR-induced lifespan extension in some lower organisms. Furthermore, the effects of DR on sirtuins (e.g. SIRT1, SIRT2, SIRT3, and SIRT5) have also been reported in mammals. However, the relationship between sirtuins and DR-associated longevity in mammals is still not clear. In addition, ageing and DR-associated changes in cellular protein acetylation have not been fully elucidated, especially in DR-aged animals. Objective: We aimed to elucidate the effect of ageing and DR on cellular protein acetylation in young and aged rats. Methods: Fischer 344 rats were subjected to DR for 7.5 or 25.5 months from 1.5 months of age. Protein acetylation status in tissues was analyzed by Western blotting, subcellular fractionation, and immuno-pull-down assay. We also analyzed the quantitative changes in some related deacetylases and an acetyltransferase. Results: Acetylation of multiple proteins in the liver of young and aged rats decreased slightly with ageing and increased markedly under DR. The results of subcellular fractionation revealed that the DR-induced increase in protein acetylation was more prominent in extranuclear proteins than in nuclear proteins, indicating that acetylation is global, but protein-specific. This was further confirmed in the results of immune-pull-down assays for mitochondrial acetylated proteins. Cellular protein acetylation is regulated by multiple factors, including various deacetylases and acetyltransferases. With regard to the possible mechanisms of DR-induced increases in protein acetylation, we observed that DR increased SIRT3 expression in the liver of young and aged rats. Expression of the mitochondrial protein acetyltransferase GCN5L1 significantly increased with ageing but did not respond to DR. Conclusions: The increased acetylation of extranuclear proteins may be involved in DR-induced anti-ageing effects including longevity. However, the mechanisms underlying the changes in protein acetylation might not result from quantitative changes in mitochondrial sirtuins and the mitochondrial protein acetyltransferase.

Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

A phase I first‐in‐human study with tefinostat – a monocyte/macrophage targeted histone deacetylase inhibitor – in patients with advanced haematological malignancies

Tefinostat (CHR-2845) is a monocyte/macrophage targeted histone deacetylase inhibitor (HDACi). This first-in-human, standard 3 + 3 dose escalating trial of oral, once daily tefinostat was conducted to determine the safety, tolerability, pharmacokinetic and pharmacodynamic profile of tefinostat in relapsed/refractory haematological diseases. Eighteen patients were enrolled at doses of 20-640 mg. Plasma concentrations of tefinostat exceeded those demonstrated to give in vitro anti-proliferative activity. Flow cytometric pharmacodynamic assays demonstrated monocyte-targeted increases in protein acetylation, without corresponding changes in lymphocytes. Dose-limiting toxicities (DLTs) were not observed and dose escalation was halted at 640 mg without identification of the maximum tolerated dose. Drug-related toxicities were largely Common Toxicity Criteria for Adverse Events grade 1/2 and included nausea, anorexia, fatigue, constipation, rash and increased blood creatinine. A patient with chronic monomyelocytic leukaemia achieved a bone marrow response, with no change in peripheral monocytes. An acute myeloid leukaemia type M2 patient showed a >50% decrease in bone marrow blasts and clearance of peripheral blasts. In conclusion, tefinostat produces monocyte-targeted HDACi activity and is well tolerated, without the DLTs, e.g. fatigue, diarrhoea, thrombocytopenia, commonly seen with non-targeted HDACi. The early signs of efficacy and absence of significant toxicity warrant further evaluation of tefinostat in larger studies. (clinicaltrials.gov identifier: NCT00820508).

Abstract 521: Mechanisms of chemoresistance: linking lysine acetylation to cell death.

Abstract Breast tumors often possess regions of hypoxia in which tumor cells must either adapt metabolically to survive or die. Some tumor cells adapt and, through a process that is poorly understood, become chemoresistant. We are interested in defining the mechanisms by which this chemoresistance develops, with focus on the role of lysine acetylation_an enigmatic protein modification thought to play a role in tuning metabolism to the available nutrient supply. We posit that changes in lysine acetylation reflect metabolic adaptation to hypoxia and modulate apoptotic-signaling pathways. The importance of protein lysine acetylation in tumor cell death is evidenced by numerous clinical cancer trials testing the tumor-killing potential of deacetylase inhibitors (e.g., Varinostat). However, the molecular mechanisms by which lysine acetylation modulates cell death are poorly understood. Using a proteomics approach to characterize acetylation dynamics under conditions of hypoxic stress in breast cancer cells, we found a striking correlation between changes in acetylation and hypoxia sensitivity. Specifically, proteome-wide increases in protein acetylation were only observed in hypoxia-sensitive cell lines, likely reflecting their attempt to adapt metabolically to hypoxia. Moreover, these experiments revealed potential networks of acetylated proteins involved in the hypoxia response, including metabolic enzymes, oncogenes (e.g., c-myc), nutrient-responsive transcription factors, and 14-3-3ζ. Our previously published data suggested that acetylation of 14-3-3ζ, a small pro-survival phospho-binding protein, led to its release from protein complexes and consequently sensitized cells to apoptosis (1). Consistent with this idea, we observed a 12-fold hypoxia-induced increase in 14-3-3ζ acetylation in the hypoxia-sensitive cells (with no detectable change in 14-3-3ζ acetylation in resistant cells). Moreover, this acetylation correlated with a shift of 14-3-3ζ from high to low molecular weight gel filtration fractions, indicating a dissociation of 14-3-3ζ from protein complexes. Proteomics efforts to characterize the 14-3-3ζ interactome in sensitive cells revealed that hypoxia triggers the release of 14-3-3ζ from proteins involved in glucose metabolism, and shifts its binding to proteins involved in autophagy and starvation metabolism, implicating 14-3-3ζ as a regulator of metabolism under these conditions. Importantly, depletion of 14-3-3ζ with siRNA (thus perturbing its network of interactions) potently sensitized all breast cancer cell lines, including the most highly resistant cells, to hypoxia. Collectively, our data suggest that lysine acetylation plays an integral part in dictating the survival of cells in hypoxia. Moreover, we propose that 14-3-3ζ, and the pathways that govern its acetylation, are promising therapeutic targets to sensitize breast cancer cells to death. Citation Format: Vajira Weerasekara, Jeff Mortenson, Lisa Heppler, Joshua Lyon Andersen. Mechanisms of chemoresistance: linking lysine acetylation to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 521. doi:10.1158/1538-7445.AM2013-521

SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Pharmacodynamic Assessment of Histone Deacetylase Inhibitors: Infrared Vibrational Spectroscopic Imaging of Protein Acetylation

Infrared spectroscopy identifies molecules by detection of vibrational patterns characteristic of molecular bonds. We apply this approach to measure protein acetylation after treatment with histone deacetylase inhibitors. The anticancer activity of histone deacetylase inhibitors (HDACi) is ascribed to the hyperacetylation of both core nucleosomal histones and nonhistone proteins critical to the maintenance of the malignant phenotype (Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Curr. Opin. Oncol. 2001, 13, 477-483; Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.; Simeoni, S.; Ragno, R. Med. Res. Rev. 2005, 25, 261-309). After incubation of the peripheral blood mononuclear cells (PBMCs) in vitro with the HDACi SNDX-275, a benzamide drug derivative, vibrational spectral changes in the methyl and methylene stretching mode regions, which reflect concentration-dependent increases in protein acetylation, were detected and quantified. We applied these metrics, based upon spectral differences, to peripheral blood mononuclear cells from patients treated in vivo with this agent. The data demonstrate a new approach to a sensitive assessment of global molecular modifications that is independent of antibodies, requires minimal cell processing, and is easily adapted to high-throughput screening.