Project description:Shotgun proteomic analysis of 3D-grown or on CAM-assay grown lung cancer cells lacking or expressing the major triglyceride lipase adipose triglyceride lipase (ATGL)
Project description:HEL92.1.7 cells in exponential growth phase were treated with 100 nM of SY-5609 for 24 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and lethal effects of treatment with a CDK7 inhibitor in secondary AML cells.
Project description:Patient-derived secondary AML cells were treated with 100 nM of SY-5609 for 24 hours, 100 nM of FHD-286 for 48 hours, or 20 µM of Tasquinimod for 48 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and lethal effects of treatment with a CDK7 inhibitor, chromatin remodeling inhibitor, or S100A8/S100A9 inhibitor or in secondary AML cells. Patient-derived de novo AML cells with MLL1 rearrangement and Menin T349M mutation were treated with 100 nM of FHD-286 or 500 nM SNDX-50469 for 48 hours to determine the global protein expression alterations that correlate with the cell cycle, growth inhibitory and/or lethal effects of treatment with a chromatin remodeling inhibitor, FHD-286, or a Menin inhibitor SNDX-50469 in MLL1 rearranged AML cells
Project description:Inhibition of adipocyte triglyceride lipase (ATGL/PNPLA2) activity in insulin resistance and non-alcoholic fatty liver disease is an attractive therapeutic target. This study evaluated the impact of Atglistatin-mediated ATGL inhibition on non-alcoholic steatohepatitis (NASH) development in diabetic and hyperlipidaemic mice. Streptozotocin-injected male mice were fed an HFD to induce NASH. We analysed liver histology, hepatic lipid content, immunohistochemistry, RNA sequencing, and serum biochemistry. Mechanistically, we treated Caco2 cells, human primary ileum-derived organoids, and HepG2 ATGL KD cells with the novel human ATGL inhibitor NG-497 and WY14643. Atglistatin reduced body and liver weights, total liver lipid content, and improved blood sugar levels consistent with improved liver enzymes, histological liver injury, and NAFLD activity scores. Mechanistically, Atglistatin reduced Cpt1a and Abca1, suggesting impaired Ppara signalling that favours hydrophilic bile acids (BA) synthesis since Cyp7a1, Cyp27a1, and Cyp2c70 were increased while Cyp8b1 was reduced. Accordingly, Intestinal lipid transporters, Abca1 and Cd36, were reduced, consistent with the reduction of liver TAG species, mostly linoleic acids. We used human-specific ATGL inhibitor NG-497 to validate the mouse findings. The NG-497-treated human primary ileum organoids and Caco2 cells showed reduced ATGL, ABCA1, FATP5, CD36, and MTTP. PPARa signalling was impaired in the ileum organoids and HepG2 treated with NG-497 and in HepG2 ATGL KD since PPARa, CPT1a, AOX, ABCA1, and CYP8B1 were reduced, and CYP7A1 increased. Inhibition of ATGL activity reduces the availability of ligands for Ppara activation leading to impaired Ppara signalling, which translate into hydrophilic BA that interferes with dietary lipid absorption, improving metabolic disturbances. The validation with the NG-497 should open a new clinical avenue for NAFLD clinical trial and treatment.
Project description:Lysosomal acid lipase (LAL) is the key enzyme of lysosomal lipid hydrolysis, which degrades cholesteryl esters (CE), triacylglycerols (TG), diacylglycerols (DG), and retinyl esters. The role of LAL in various cellular processes has mostly been studied in LAL-deficient (Lal-/-) mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 (L1) and Lalistat-2 (L2). Here, we show that pharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis and neutral TG hydrolase (TGH) and CE hydrolase (CEH) activities in mature adipocytes, indicating that L1 and L2 inhibit other lipid hydrolases apart from LAL. Since adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes that degrade cytosolic TG and CE, respectively, at neutral pH, we hypothesized that L1 and L2 also inhibit ATGL and/or HSL through off-target effects. In fact, both inhibitors drastically reduced neutral CEH activity in cells overexpressing mouse and human HSL and neutral TGH activity in cells overexpressing mouse and human ATGL, albeit to a lesser extent. By performing serine hydrolase-specific activity-based labeling in combination with quantitative proteomics, we confirmed that L2 inhibits HSL and other lipid hydrolases, whereas L1 treatment results in less pronounced inhibition of neutral lipid hydrolases. These results demonstrate that commonly used concentrations of L2 (and L1) are not suitable for investigating the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy.
Project description:Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins and is involved in many physiological processes. To investigate the role of adipose HuR, we generate adipose-specific HuR knockout (HuRAKO) mice. As compared with control mice, HuRAKO mice show obesity when induced with a high-fat diet, along with insulin resistance, glucose intolerance, hypercholesterolemia and increased inflammation in adipose tissue. The obesity of HuRAKO mice is attributed to adipocyte hypertrophy in white adipose tissue due to decreased expression of adipose triglyceride lipase (ATGL), a critical lipase involved in lipolysis. HuR positively regulates ATGL expression by promoting the mRNA stability and translation of ATGL. Consistently, the expression of HuR in adipose tissue is reduced in obese humans, which is associated with reduced ATGL expression. This study suggests that adipose HuR may be a critical regulator of ATGL expression and lipolysis and thereby controls obesity and metabolic syndrome.
Project description:This study examines and compares the protein content in conditioned media collected from neural cell types generated from human pluripotent stem cells. Conditioned media was prepared for 48 hours at a final endpoint of differentiation day 12. Both groups are from parental line WTC11 and cultured as a monolayer on matrigel. Both groups contain a transgene cassette for doxycycline-inducible expression of sox9 and nfia. Doxycycline was only included in the iAstro groups, whereas it was omitted in the neural progenitor cell groups.
Project description:Lipid metabolism plays a central role in prostate cancer. To date, the major focus on prostate cancer lipid metabolism has centered on the roles of de novo lipogenesis and lipid uptake with little consideration for how cancer cells access these lipids once they are created or taken up and stored. Analysis of patient-derived phosphoproteomics data identified adipose triglyceride lipase (ATGL), a rate-limiting enzyme in the breakdown of triglycerides and previously suspected tumor suppressor, as a target of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-AMP-dependent protein kinase (AMPK) signaling that, conversely, promotes castration-resistant prostate cancer (CRPC) progression. Phosphorylation of ATGL by CAMKK2-AMPK signaling increased ATGL’s lipase activity, cancer cell proliferation, migration, and invasion. Shotgun lipidomics and imaging mass spectrometry demonstrated ATGL’s profound regulation of prostate cancer lipid metabolism in vitro and in vivo, remodeling membrane composition. Targeting ATGL using molecular, genetic, and/or pharmacological approaches impaired the growth of human and murine prostate cancer cells in culture and xenograft mouse models of CRPC as well as organoid models. The efficacy of ATGL inhibition occurs in a glucose concentration-dependent manner. Accordingly, pharmacological inhibition or depletion of ATGL induced metabolic plasticity with a shift towards glycolysis, which could be exploited therapeutically by co-targeting both metabolic pathways. Together, these data nominate ATGL and intracellular lipolysis as a potential therapeutic target for the treatment of CRPC and provide insights for future combination therapies.
Project description:We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (ID), and that NUDT21-encoded CFIm25 regulates the protein levels of at least one dose-sensitive, ID-associated protein: MeCP2 (Gennarino et al., 2015). However, the patients’ CNVs also spanned multiple other genes raising the possibility that loss or gain of these other genes caused their symptoms. To determine if reduced NUDT21 function alone is sufficient to cause disease, we generated Nudt21 heterozygous null mice to mimic the human state of reduced expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human embryonic stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which are associated with intellectual disability and whose dysregulation is likely contributing to disease symptoms. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.
Project description:Obesity is a worldwide epidemic associated with increased risk and progression of colon cancer. Here, we aimed to determine the role of adipose triglyceride lipase (ATGL), responsible for intracellular lipid droplet (LDs) utilization, in obesity driven colonic tumorigenesis. In local colon cancer patients, significantly increased ATGL levels in tumor tissue, compared to controls, were augmented in obese individuals. Elevated ATGL levels in human colon cancer cells (CCC) relative to non-transformed were augmented by an obesity mediator, oleic acid (OA). In CCC and colonospheres, enriched in colon cancer stem cells (CCSC), inhibition of ATGL prevented LDs utilization and inhibited OA-stimulated growth through retinoblastoma-mediated cell-cycle arrest. Further, transcriptomic analysis of CCC, with inhibited ATGL, revealed targeted pathways driving tumorigenesis and high-fat-diet obesity facilitated tumorigenic pathways. Inhibition of ATGL in colonospheres revealed targeted pathways in human colonic tumor crypt base cells (enriched in CCSC) derived from colon cancer patients. In CCC and colonospheres, we validated selected transcripts targeted by ATGL inhibition, some with emerging roles in colonic tumorigeneses (ATG2B, PCK2, PGAM1, SPTLC2, IGFBP1, ABCC3) and others with established roles (MYC, MUC2). These findings demonstrate obesity-promoted, ATGL-mediated colonic tumorigenesis and establishes therapeutic significance of ATGL in obesity reinforced colon cancer progression.