Julia Petschnigg

Obese Yeast: Triglyceride Lipolysis Is Functionally Conserved from Mammals to Yeast

Storage and degradation of triglycerides are essential processes to ensure energy homeostasis and availability of precursors for membrane lipid synthesis. Recent evidence suggests that an emerging class of enzymes containing a conserved patatin domain are centrally important players in lipid degradation. Here we describe the identification and characterization of a major triglyceride lipase of the adipose triglyceride lipase/Brummer family, Tgl4, in the yeast Saccharomyces cerevisiae. Elimination of Tgl4 in a tgl3 background led to fat yeast, rendering growing cells unable to degrade triglycerides. Tgl4 and Tgl3 lipases localized to lipid droplets, independent of each other. Serine 315 in the GXSXG lipase active site consensus sequence of the patatin domain of Tgl4 is essential for catalytic activity. Mouse adipose triglyceride lipase (which also contains a patatin domain but is otherwise highly divergent in primary structure from any yeast protein) localized to lipid droplets when expressed in yeast, and significantly restored triglyceride breakdown in tgl4 mutants in vivo. Our data identify yeast Tgl4 as a functional ortholog of mammalian adipose triglyceride lipase.

Cdk1/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression.

Triacylglycerols (TGs) serve essential cellular functions as reservoirs for energy substrates (fatty acids) and membrane lipid precursors (diacylglycerols and fatty acids). Here we show that the major yeast TG lipase Tgl4, the functional ortholog of murine adipose TG lipase ATGL, is phosphorylated and activated by cyclin-dependent kinase 1 (Cdk1/Cdc28). Phospho-Tgl4-catalyzed lipolysis contributes to early bud formation in late G1 phase of the cell cycle. Conversely, lack of lipolysis delays bud formation and cell-cycle progression. In the absence of beta-oxidation, lipolysis-derived metabolites are thus required to support cellular growth. TG homeostasis is the only metabolic process identified as yet that is directly regulated by Cdk1/Cdc28-dependent phosphorylation of key anabolic and catabolic enzymes, highlighting the importance of FA storage and mobilization during the cell cycle. Our data provide evidence for a direct link between cell-cycle-regulatory kinases and TG degradation and suggest a general mechanism for coordinating membrane synthesis with cell-cycle progression.

Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast.

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells

Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin–based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non–small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.

Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly.

We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.

Systematic Identification of Oncogenic EGFR Interaction Partners

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) that—once activated upon ligand binding—leads to receptor dimerization, recruitment of protein complexes, and activation of multiple signaling cascades. The EGFR is frequently overexpressed or mutated in various cancers leading to aberrant signaling and tumor growth. Hence, identification of interaction partners that bind to mutated EGFR can help identify novel targets for drug discovery. Here, we used a systematic approach to identify novel proteins that are involved in cancerous EGFR signaling. Using a combination of high-content imaging and a mammalian membrane two-hybrid protein–protein interaction method, we identified eight novel interaction partners of EGFR, of which half strongly interacted with oncogenic, hyperactive EGFR variants. One of these, transforming acidic coiled-coil proteins (TACC) 3, stabilizes EGFR on the cell surface, which results in an increase in downstream signaling via the mitogen-activated protein kinase and AKT pathway. Depletion of TACC3 from cells using small hairpin RNA (shRNA) knockdown or small-molecule targeting reduced mitogenic signaling in non-small cell lung cancer cell lines, suggesting that targeting TACC3 has potential as a new therapeutic approach for non-small cell lung cancer.

GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling

High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).

Abstract A20: Insulin receptor signaling in mammary epithelial cells

Obesity has recently emerged as a major adverse factor in the prevalence and severity of many types of cancers, including breast cancer (BC). Obesity is often accompanied by hyperinsulinemia and the development of type 2 diabetes. Elevated circulating insulin levels are thought to mediate some of the adverse prognostic effects of obesity in BC (Goodwin et al., 2002). Insulin receptor (IR) is expressed in many human BCs and the activation of the IR/receptor tyrosine kinase signaling pathway is characteristic of most BCs (Mulligan et al., 2007). We hypothesize that ectopic expression of IR in BCs sensitizes BC cells to the action of insulin, leading to signaling events specific to epithelial cells, functionally distinct from the insulin responses in physiological insulin-target tissues. To further understand the role of IR in BC we have implemented the recently developed Mammalian Membrane Two Hybrid (MaMTH) (Petschnigg et al., 2014) screen to identify IR interactors in human mammary epithelial MCF10A cells. The ongoing screen has to date identified 99 IR protein-protein interactions (PPIs), 51 of which are insulin-dependent. Coimmunoprecipitation and shRNA knockdown analyses are being used to validate the PPIs and provide insight into the functional significance of IR associations for growth and proliferation of MCF10A cells. Ultimately, the epithelial cell IR-interactome is meant to provide an insight into the insulin contribution to the activation of RTK signaling in BC, and provide a roadmap for further exploration of IR signaling in cancer. Citation Format: Yekaterina Poloz, Mariam Z. El Sheikh, Julia Petschnigg, Bella Groisman, Max Kotlyar, Igor Jurisica, Igor Stagljar, Vuk Stambolic. Insulin receptor signaling in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A20.