Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids

Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity.

Kinetic reconstruction and analysis of sphingolipid metabolism

Vassily Hatzimanikatis, Howard Riezman, Georgios Savoglidis

Lipids are major constituents of the cell. They are responsible for major properties of the cellular membranes: hydrophobicity, selective permeability and being the scaffold of signaling proteins. Many diseases are associated with alterations in the lipid distribution in the cell and the composition of membrane domains. Metabolic syndrome, obesity, atherosclerosis, as well as Alzheimer’s, Huntington’s diseases and cancer, have an impact in the levels of lipids, with observed alterations in their concentrations compared to the healthy state. Applying computational techniques along with systematic modeling of lipid metabolism can provide insights that can guide biomedical research and develop potential strategies for prevention and cure. In the present study we developed a comprehensive model of the sphingolipid biosynthesis in the yeast Saccharomyces cerevisiae. Sphingolipids are one of the four major lipid categories, along with (glycero)phospholipids, sterols and fatty acids synthesized in the yeast S. cerevisiae. The importance of sphingolipids present in any higher eukaryote has been demonstrated in many recent studies. For this study, S. cerevisiae has been chosen as a model organism due to its high homology of cellular processes with mammalian cells. The developed model will be an essential part towards the construction of a detailed kinetic model of the whole lipidome of the cell. We first constructed a stoichiometric model that contains all the currently known reactions for the biosynthesis of ceramides and complex sphingolipids in the yeast. Additionally, we have accounted for all five reported hydroxylation states, along with the reactions synthesizing the necessary precursor metabolites from other lipid pathways (i.e. palmitate-CoA from fatty acid synthesis and phosphatidylinositol from the phospholipids metabolism). We next developed a kinetic model and we used a large number of lipidomic measurements of wild type yeast to consistently calibrate our model. Curation of the kinetic information of the model came from the comprehensive mining of references for operation of the enzymes as well as ranges of kinetic parameters from online databases. These datasets created the pool for a sampling technique that accounts for the uncertainty in the parameters of the model. This lead to a robust dynamic model, containing mass balances for all the components of the biochemical network as well as terms that accounted for the dillution of these molecules in the cell due to growth. We performed a thorough kinetic analysis of the system by examining the impact of different assumptions in enzyme operation on the levels of ceramides and complex sphingolipids (e.g. substrate competition, (un)competitive inhibition). By applying the principles of Metabolic Control Analysis (MCA) we were able to quantify the effect of enzyme activities on the lipid profiles and we identified enzymes of the biochemical reaction network as efficient targets for metabolic engineering towards a desired state. We also applied a reverse engineering method and we were able to identify enzyme perturbations responsible for an observed altered state compared to the wild type cellular lipidomic profile. Such an approach could lead to the identification of genetic mutations by imploring the information containd within metabolomic measurements. Although we demonstrate the application of the method in models of lipid metabolism it is possible to be applied to biochemical systems of various parts of metabolism and sizes creating a modular platform for kinetic analysis of cellular operations.

2014

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Howard Riezman

Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected.(jlr) We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules.-Loizides-Mangold, U., F. P. A. David, V. J. Nesatyy, T. Kinoshita, and H. Riezman. Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels. J. Lipid Res. 2012. 53: 1522-1534.

2012

Regulation of Endoplasmic Reticulum Architecture by Protein S-Palmitoylation

Patrick Alain Sandoz

The endoplasmic reticulum (ER) is the largest organelle in mammalian cells. More than 50 years ago, its morphology was already described as consisting of two major compartments: the rough ER and the smooth ER, which both fulfil precise and distinct functions to sustain the cellular metabolism. The rough ER localizes in the perinuclear region and is composed of packed membrane sheets with studded ribosomes. The smooth ER extends to the cell periphery as a continuous tubular membrane meshwork. Impairment of the ER structure is related to multiple diseases, in particular psycho-motor degenerations such as hereditary spastic paraplegia. The current model of the ER architecture suggests that specific ER proteins, called ER-shaping proteins, generate and maintain the ER morphology. Different families of ER-shaping proteins support either the curvature or the flatness of the membrane and the exact ER morphology depends on their local concentrations. Although the structural role of these proteins has been extensively characterized, the mechanism of their regulation by the cell is mostly unknown. Moreover, the process has to be very dynamic, as the ER membrane morphology is rapidly interconverted. Our work has discovered that several ER-shaping proteins are modified by a post-translational modification named S-palmitoylation. S-palmitoylation is the covalent but reversible attachment of a 16 carbons chain fatty acid to a cysteine residue of a protein. Interestingly, both ER-sheet and ER-tubule promoting proteins were modified. We observed that these palmitoylated ER-shaping proteins are all controlled by the same palmitoyl-acyltransferase: DHHC6. Thus, our results suggest that DHHC6 is a master regulator of the ER architecture. We also demonstrated that palmitoylation stabilizes the lifetime of one specific substrate: CLIMP-63, which induces ER-sheet proliferation. Transient expression of DHHC6 generated prominent ER-sheets, which were dependent on CLIMP-63's presence and palmitoylation. On the contrary, the absence of DHHC6 diminished the size of the rough ER. Finally, to study protein palmitoylation, we developed a novel assay which was able to quantify the fraction of palmitoylated protein at steady state. Taken together, our data highlight the first general mechanism that mediates the ER architecture. Our future work will focus on understanding how and when the cell uses palmitoylation of ER-shaping proteins and clarifying the effectors of this signalling pathway.