Browsing by Author "Ven, Katharina"

Lipid droplets (LDs) are ubiquitous intracellular storage organelles, consisting of a core of energy rich neutral lipids surrounded by a phospholipid monolayer. Research in the past decade has expanded the view on LDs from simple, passive cytosolic inclusions to dynamic organelles which play an important role in many cellular processes. Furthermore, there is mounting evidence for links between LD biology and human pathologies, such as metabolic disorders, non-alcoholic fatty liver disease and cardiovascular diseases. Thus, understanding the basic biology of LD formation is crucial. LD biogenesis is thought to occur in the microdomains of endoplasmic reticulum (ER), due to the accumulation of neutral lipids between the two leaflets of the ER bilayer before budding into the cytosol. Many proteins are involved in this early formation, but no single indispensable protein has been discovered. After assembly, these early LDs grow through lipid deposition from the ER, and with lipid synthesis on the droplet monolayer. During LD growth, LDs are thought to retain connection to the ER. A protein important for LD biogenesis is seipin. This oligomeric ER protein has been found to localize at contact sites between the ER and LDs. Mutations in seipin give rise to three distinct diseases in humans; BSCL2, seipinopathy and Celia’s encephalopathy. The role of seipin in the formation of LDs and the pathogenesis of these diseases is still unknown. Work from numerous model systems has shown seipin to be important for LD biogenesis and adipocyte differentiation. LD formation is a complex process which is still poorly understood, and seipin likely collaborates with other proteins during LD assembly. In this thesis, APEX2-mediated proteome mapping combined with LC-MS/MS, is set up to identify proteins involved in LD biogenesis. In this technology, an engineered ascorbate peroxidase, APEX2, is genetically inserted to the intracellular region of interest where it rapidly biotinylates nearby endogenous proteins upon exposure to biotin-phenol and hydrogen peroxide. Biotinylated proteins can then be enriched by using streptavidin beads and identified with a mass spectrometry. The aim using this technology is to unravel new interaction partners of seipin and proteins important for LD formation, which is a crucial step for understanding LD formation and diseases related to it.