Mitochondrial GCN5L1 acts as a novel regulator for iron homeostasis to promote sorafenib sensitivity in hepatocellular carcinoma.

BACKGROUND: Sorafenib resistance is becoming increasingly common and disadvantageous for hepatocellular carcinoma (HCC) treatment. Ferroptosis is an iron dependent programmed cell death underlying the mechanism of sorafenib. Iron is crucial for synthesis of cofactors essential to mitochondrial enzymes and necessary for HCC proliferation, while mitochondrial iron overload and oxidative stress are associated with sorafenib induced ferroptosis. However, the crosstalk among iron homeostasis and sorafenib resistance is unclear. METHODS: We conducted bioinformatics analysis of sorafenib treated HCC datasets to analyze GCN5L1 and iron related gene expression with sorafenib resistance. GCN5L1 deleted HCC cell lines were generated by CRISPR technology. Sorafenib resistant HCC cell line was established to validate dataset analysis and evaluate the effect of potential target. RESULTS: We identified GCN5L1, a regulator of mitochondrial acetylation, as a modulator in sorafenib-induced ferroptosis via affecting mitochondrial iron homeostasis. GCN5L1 deficiency significantly increased sorafenib sensitivity in HCC cells by down-regulating mitochondrial iron transporters CISD1 expression to induce iron accumulation. Mitochondrial iron accumulation leads to an acceleration in cellular and lipid ROS. Sorafenib resistance is related to CISD1 overexpression to release mitochondrial iron and maintaining mitochondrial homeostasis. We combined CISD1 inhibitor NL-1 with sorafenib, which significantly enhanced sorafenib-induced ferroptosis by promoting mitochondrial iron accumulation and lipid peroxidation. The combination of NL-1 with sorafenib enhanced sorafenib efficacy in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that GCN5L1/CISD1 axis is crucial for sorafenib resistance and would be a potential therapeutic strategy for sorafenib resistant HCC.

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

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

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

BACKGROUND: The 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. METHOD: GCN5L1 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. RESULTS: Knocking 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. CONCLUSIONS: In 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.

GCN5L1-mediated acetylation prevents Rictor degradation in cardiac cells after hypoxic stress.

Cardiomyocyte apoptosis and cardiac fibrosis are the leading causes of mortality in patients with ischemic heart disease. As such, these processes represent potential therapeutic targets to treat heart failure resulting from ischemic insult. We previously demonstrated that the mitochondrial acetyltransferase protein GCN5L1 regulates cardiomyocyte cytoprotective signaling in ischemia-reperfusion injury in vivo and hypoxia-reoxygenation injury in vitro. The current study investigated the mechanism underlying GCN5L1-mediated regulation of the Akt/mTORC2 cardioprotective signaling pathway. Rictor protein levels in cardiac tissues from human ischemic heart disease patients were significantly decreased relative to non-ischemic controls. Rictor protein levels were similarly decreased in cardiac AC16 cells following hypoxic stress, while mRNA levels remained unchanged. The reduction in Rictor protein levels after hypoxia was enhanced by the knockdown of GCN5L1, and was blocked by GCN5L1 overexpression. These findings correlated with changes in Rictor lysine acetylation, which were mediated by GCN5L1 acetyltransferase activity. Rictor degradation was regulated by proteasomal activity, which was antagonized by increased Rictor acetylation. Finally, we found that GCN5L1 knockdown restricted cytoprotective Akt signaling, in conjunction with decreased mTOR abundance and activity. In summary, these studies suggest that GCN5L1 promotes cardioprotective Akt/mTORC2 signaling by maintaining Rictor protein levels through enhanced lysine acetylation.

Fibroblast-to-cardiomyocyte lactate shuttle modulates hypertensive cardiac remodelling.

BACKGROUND: Cardiac fibroblasts (CFs) and cardiomyocytes are the major cell populations in the heart. CFs not only support cardiomyocytes by producing extracellular matrix (ECM) but also assimilate myocardial nutrient metabolism. Recent studies suggest that the classical intercellular lactate shuttle may function in the heart, with lactate transported from CFs to cardiomyocytes. However, the underlying mechanisms regarding the generation and delivery of lactate from CFs to cardiomyocytes have yet to be explored. RESULTS: In this study, we found that angiotensin II (Ang II) induced CFs differentiation into myofibroblasts that, driven by cell metabolism, then underwent a shift from oxidative phosphorylation to aerobic glycolysis. During this metabolic conversion, the expression of amino acid synthesis 5-like 1 (GCN5L1) was upregulated and bound to and acetylated mitochondrial pyruvate carrier 2 (MPC2) at lysine residue 19. Hyperacetylation of MPC2k19 disrupted mitochondrial pyruvate uptake and mitochondrial respiration. GCN5L1 ablation downregulated MPC2K19 acetylation, stimulated mitochondrial pyruvate metabolism, and inhibited glycolysis and lactate accumulation. In addition, myofibroblast-specific GCN5L1-knockout mice (GCN5L1fl/fl: Periostin-Cre) showed reduced myocardial hypertrophy and collagen content in the myocardium. Moreover, cardiomyocyte-specific monocarboxylate transporter 1 (MCT1)-knockout mice (MCT1fl/fl: Myh6-Cre) exhibited blocked shuttling of lactate from CFs to cardiomyocytes and attenuated Ang II-induced cardiac hypertrophy. CONCLUSIONS: Our findings suggest that GCN5L1-MPC2 signalling pathway alters metabolic patterns, and blocking MCT1 interrupts the fibroblast-to-cardiomyocyte lactate shuttle, which may attenuate cardiac remodelling in hypertension.

Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue.

GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in a number of key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.

Mitochondrial general control of amino acid synthesis 5 like 1 promotes nonalcoholic steatohepatitis development through ferroptosis-induced formation of neutrophil extracellular traps.

BACKGROUND: Mitochondria play central roles in metabolic diseases including nonalcoholic steatohepatitis (NASH). However, how mitochondria regulate NASH progression remains largely unknown. Our previous findings demonstrate that mitochondrial general control of amino acid synthesis 5 like 1 (GCN5L1) is associated with mitochondrial metabolism. Nevertheless, the roles of GCN5L1 in NASH are unclear. AIMS AND METHODS: The GCN5L1 expression was detected in the fatty livers of NASH patients and animals. Hepatocyte-specific GCN5L1 deficiency or overexpression mice were used to induce NASH models by feeding with a high-fat/high-cholesterol or methionine-choline deficient diet. The molecular mechanisms underlying GCN5L1-regulated NASH were further explored and verified in mice. RESULTS AND CONCLUSIONS: GCN5L1 expression was increased in NASH patients. Upregulated GCN5L1 level was also illustrated in NASH mice. Mice with hepatocyte-specific GCN5L1 conditional knockout improved the inflammatory response compared to GCN5L1flox/flox mice. However, overexpression of mitochondrial GCN5L1 augmented the inflammatory response. Mechanically, GCN5L1 acetylated CypD and enhanced its binding with ATP5B, which induced the opening of mitochondrial permeability transition pores and the release of mitochondrial ROS into the cytoplasm. The increased ROS promoted ferroptosis of hepatocytes and induced accumulation of high mobility group box 1 in the microenvironment, which recruited neutrophils and induced the generation of neutrophil extracellular traps (NETs). NETs block impaired GCN5L1-induced NASH progression. Furthermore, the upregulation of GCN5L1 in NASH was contributed by lipid overload-induced endoplasmic reticulum stress. Together, mitochondrial GCN5L1 has a vital function in promoting NASH progression by regulating oxidative metabolism and the hepatic inflammatory microenvironment. Thus, GCN5L1 might be a potential intervention target in NASH treatment.

GCN5L1-mediated TFAM acetylation at K76 participates in mitochondrial biogenesis in acute kidney injury.

BACKGROUND: Mitochondrial dysfunction is an important pathogenic event in acute kidney injury (AKI). GCN5L1 is a specific acetyltransferase in mitochondria, which regulates glucose and fatty acid metabolism. However, the role of GCN5L1 in mitochondrial dysfunction and the pathogenesis of ischemic AKI are not fully understood. METHODS: The protein level of GCN5L1 was detected by western blot assay. Acetylated proteomics was used to explore the level of acetylated TFAM. Duolink proximity ligation assay and co-immunoprecipitation were used to detect the interaction of TFAM and translocase of outer membrane 70 (TOM70). mtDNA copy number, the expression of mitochondrial electron transport chain complexes, the number and morphology of mitochondria were measured. The renal injury of AKI mice was reflected by the levels of creatinine and urea nitrogen and the pathological changes of renal tissue. RESULTS: We showed that GCN5L1 was highly expressed in vivo and in vitro and renal tubules specific knockdown of GCN5L1 could effectively attenuate AKI-induced mitochondrial impairment. Besides, acetylated proteomics revealed that acetylated TFAM was significantly upregulated in AKI mice kidney, which reminded us that TFAM might be an acetylating substrate of GCN5L1. Mechanistically, we evidenced that GCN5L1 could acetylate TFAM at its K76 site and subsequently inhibited its binding to TOM70, thereby reducing TFAM import into mitochondria and mitochondrial biogenesis. Clinically, GCN5L1 and acetylated TFAM were positively correlated with disease severity (all p < 0.05). CONCLUSIONS: In sum, these data demonstrated an unrecognized regulating mechanism of GCN5L1 on TFAM acetylation and its intracellular trafficking, and a potential intervening target for AKI associated mitochondrial disorders as well.

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.

BACKGROUND: Glutaminolysis 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. METHODS: Cell 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. RESULTS: Hepatic 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. CONCLUSIONS: Our 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.

GPER-dependent estrogen signaling increases cardiac GCN5L1 expression.

Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, but the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we describe a significant difference in GCN5L1 levels between male and female mouse hearts, and in the hearts of women between post- and premenopausal age. We further find that estrogen drives GCN5L1 expression in a cardiac cell line and uses pharmacological approaches to determine the mechanism to be G protein-coupled estrogen receptor (GPER) activation, via translational regulation.NEW & NOTEWORTHY We demonstrate here for the first time that mitochondrial protein acetylation is increased in female hearts, associated with an increase in GCN5L1 levels through a GPER-dependent mechanism. These findings reveal a new potential mediator of divergent cardiac mitochondrial function between men and women.

BLOC1S1/GCN5L1/BORCS1 is a critical mediator for the initiation of autolysosomal tubulation.

The mechanisms orchestrating recycling of lysosomes through autophagic lysosome reformation (ALR) is incompletely understood. Previous data show that genetic depletion of BLOC1S1/GCN5L1/BORCS1 increases autolysosome (AL) accumulation. We postulated that this phenotype may manifest due to perturbed ALR. We explored this in control and bloc1s1 liver-specific knockout (LKO) mouse hepatocytes, showing that in response to nutrient-deprivation LKO's fail to initiate ALR due to blunted lysosomal tubulation. As kinesin motor proteins and the intracellular cytoskeleton are requirements for tubular formation from ALs, we explored the interaction of BLOC1S1 with motor proteins and cytoskeletal factors. BLOC1S1 interacts with the ARL8B-KIF5B (GTPase and kinesin motor protein) complex to recruit KIF5B to ALs. Furthermore, BLOC1S1 interacts with the actin nucleation promoting factor WHAMM, which is an essential structural protein in the initiation of lysosomal tubulation (LT). Interestingly, the genetic reintroduction of BLOC1S1 rescues LT in LKO hepatocytes, but not when KIF5B is concurrently depleted. Finally, given the central role of MTORC1 signaling in ALR initiation, it was interesting that MTORC1 activity was increased despite the absence of LT in LKO hepatocytes. Concurrently, inhibition of MTORC1 abolished BLOC1S1 reconstitution-mediated rescue of LT in LKO hepatocytes. Taken together these data demonstrate that the functional interaction of BLOC1S1 with the kinesin binding complex and the actin cytoskeleton are a requirement for LT which, in parallel with MTORC1 signaling, initiate lysosome recycling via ALR.Abbreviations: 3-MA: 3-methyladenine; AL: autolysosome; ALR: autophagic lysosome reformation; ARL8B: ADP-ribosylation factor-like protein 8B; ARPC2: actin related protein 2/3 complex, subunit 2; ATAT1/αTAT1: alpha tubulin acetyltransferase 1; AVd: autophagic vacuoles, degradative; BLOC1S1/GCN5L1: biogenesis of lysosomal organelles complex-1, subunit 1; CQ: chloroquine; KIF5B: kinesin family member 5B; KLC1: kinesin light chain 1; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC3B-I: cytosolic form of LC3B; LC3B-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; LKO: liver-specific knockout; LIs: lysosome inhibitors; LT: lysosomal tubulation; Ly: lysosome; MTORC1: mechanistic target of rapamycin kinase complex 1; PLEKHM2/SKIP: pleckstrin homology domain containing, family M (with RUN domain) member 2; Snapin: SNAP-associated protein; SQSTM1/p62: sequestosome 1; SVPs: synaptic vesicle precursors; TFEB: transcription Factor EB; TFE3: transcription factor E3; WHAMM: WAS protein homolog associated with actin, golgi membranes and microtubules.

Increased fatty acid oxidation enzyme activity in the hearts of mice fed a high fat diet does not correlate with improved cardiac contractile function.

Changes in the acetylation status of mitochondrial proteins have been linked to the development of metabolic dysfunction in a number of tissues. Increased lysine acetylation has been reported in the hearts of obese mice, and is associated with changes in fuel metabolism, redox status, and mitochondrial oxidative phosphorylation. In this study, we examined whether diet-induced changes in the acetylation of mitochondrial acyl-CoA dehydrogenases affected fatty acid oxidation enzyme activity and contractile function in the obese mouse heart. Exposure to a long-term high fat diet in wildtype mice led to the hyperacetylation of short- and long-chain acyl-CoA dehydrogenases SCAD and LCAD, which correlated with their increased enzymatic activity in vitro. Cardiomyocyte-specific deletion of the mitochondrial acetyltransferase-related protein GCN5L1 prevented both the hyperacetylation and increased activity of these enzymes under the same conditions of dietary excess. Despite the potential for increased cardiac fatty acid oxidation activity, wildtype mice did not display any increase in cardiac contractility following exposure to a high fat diet. We conclude that the potential energetic benefits of elevated fatty acid oxidation activity are not sufficient to counter the various deleterious effects of a high fat diet on cardiac function.

GCN5L1 controls renal lipotoxicity through regulating acetylation of fatty acid oxidation enzymes.

Dyslipidemia is a common risk factor of chronic kidney disease (CKD). Current notion suggests that insufficient intracellular fatty acid oxidation (FAO) and subsequently enhanced fatty acid esterification within renal resident cells, a process termed as renal lipotoxicity, is the key pathogenic event responsible for dyslipidemia-induced kidney injury. However, the detailed mechanism is not fully elucidated. Recently, accumulating data indicated that acetylation modification is an important regulating manner for both mitochondrial function and energy metabolism, while whether acetylation modification is involved in renal lipotoxicity is of little known. In the present study, the expression level of global lysine acetylation was detected by immunohistochemistry in high-fat diet mice and western blot in palmitic acid (PA) stimulated HK-2 cells. The acetylation levels of long-chain acyl-CoA dehydrogenases (LCAD) and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) were measured by immunoprecipitation. And a multifunction microplate reader was applied to detect FAO rate, triglyceride and acyl-CoA contents, and the enzyme activities, with cellular lipid accumulation identified by Oil Red O staining. We evidenced the acetylation levels of LCAD and ß-HAD that were enhanced, which led to decreased enzymatic activities and impaired FAO rate. Furthermore, renal protein hyperacetylation induced by lipid overload was associated with increased expression of GCN5L1. And the silence of GCN5L1 in tubular epithelial cells resulted in deacetylation and activation of LCAD and ß-HAD. Finally, excess lipids induced lipotoxicity and epithelial-mesenchymal transition (EMT) were ameliorated by GCN5L1 suppression, suggesting GCN5L1-mediated mitochondrial LCAD and ß-HAD acetylation might be a key pathogenic event underlying excess lipids induced FAO impairment.

Loss of GCN5L1 in cardiac cells disrupts glucose metabolism and promotes cell death via reduced Akt/mTORC2 signaling.

GCN5L1 regulates protein acetylation and mitochondrial energy metabolism in diverse cell types. In the heart, loss of GCN5L1 sensitizes the myocardium to injury from exposure to nutritional excess and ischemia/reperfusion injury. This phenotype is associated with the reduced acetylation of metabolic enzymes and elevated mitochondrial reactive oxygen species (ROS) generation, although the direct molecular targets of GCN5L1 remain largely unknown. In this study, we sought to determine the mechanism by which GCN5L1 impacts energy substrate utilization and mitochondrial health. We find that hypoxia and reoxygenation (H/R) leads to a reduction in cell viability and Akt phosphorylation in GCN5L1 knockdown AC16 cardiomyocytes, in parallel with elevated glucose utilization and impaired fatty acid use. We demonstrate that glycolysis is uncoupled from glucose oxidation under normoxic conditions in GCN5L1-depleted cells. We show that GCN5L1 directly binds to the Akt-activating mTORC2 component Rictor, and that loss of Rictor acetylation is evident in GCN5L1 knockdown cells. Finally, we show that restoring Rictor acetylation in GCN5L1-depleted cells reduces mitochondrial ROS generation and increases cell survival in response to H/R. These studies suggest that GCN5L1 may play a central role in energy substrate metabolism and cell survival via the regulation of Akt/mTORC2 signaling.

Loss of GCN5L1 in cardiac cells limits mitochondrial respiratory capacity under hyperglycemic conditions.

The mitochondrial acetyltransferase-related protein GCN5L1 controls the activity of fuel substrate metabolism enzymes in several tissues. While previous studies have demonstrated that GCN5L1 regulates fatty acid oxidation in the prediabetic heart, our understanding of its role in overt diabetes is not fully developed. In this study, we examined how hyperglycemic conditions regulate GCN5L1 expression in cardiac tissues, and modeled the subsequent effect in cardiac cells in vitro. We show that GCN5L1 abundance is significantly reduced under diabetic conditions in vivo, which correlated with reduced acetylation of known GCN5L1 fuel metabolism substrate enzymes. Treatment of cardiac cells with high glucose reduced Gcn5l1 expression in vitro, while expression of the counteracting deacetylase enzyme, Sirt3, was unchanged. Finally, we show that genetic depletion of GCN5L1 in H9c2 cells leads to reduced mitochondrial oxidative capacity under high glucose conditions. These data suggest that GCN5L1 expression is highly responsive to changes in cellular glucose levels, and that loss of GCN5L1 activity under hyperglycemic conditions impairs cardiac energy metabolism.

Cardiac-specific deletion of GCN5L1 restricts recovery from ischemia-reperfusion injury.

GCN5L1 regulates mitochondrial protein acetylation, cellular bioenergetics, reactive oxygen species (ROS) generation, and organelle positioning in a number of diverse cell types. However, the functional role of GCN5L1 in the heart is currently unknown. As many of the factors regulated by GCN5L1 play a major role in ischemia-reperfusion (I/R) injury, we sought to determine if GCN5L1 is an important nexus in the response to cardiac ischemic stress. Deletion of GCN5L1 in cardiomyocytes resulted in impaired myocardial post-ischemic function and increased infarct development in isolated work-performing hearts. GCN5L1 knockout hearts displayed hallmarks of ROS damage, and scavenging of ROS restored cardiac function and reduced infarct volume in vivo. GCN5L1 knockdown in cardiac-derived AC16 cells was associated with reduced activation of the pro-survival MAP kinase ERK1/2, which was also reversed by ROS scavenging, leading to restored cell viability. We therefore conclude that GCN5L1 activity provides an important protection against I/R induced, ROS-mediated damage in the ischemic heart.

The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.