In Vivo Profiling and Visualization of Cellular Protein–Lipid Interactions Using Bifunctional Fatty Acids

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

“…The difference between binding energies of SMS1 to PC and PE calculated by Zhang et al (90 kJ/mol) suggests that the lat ter mechanism applies (39). However, photoaffinity label ing experiments with diazirine-containing phospholipid analogs (40) combined with a thorough analysis of enzyme kinetics will be necessary to distinguish the above two mechanisms. The latter will be a challenging task [see e.g., Mina et al (41)], because for a bifunctional enzyme like SMS2, it involves systematic variation of the concentra tions of three different substrates (i.e., ceramide, PC, and PE) when only the forward reactions are considered.…”

A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7

“…All synthetic compounds were purified by thin layer chromatography to a high degree (purity >98%) and their structures were confirmed by 1H and 13C NMR and electrospray-ionisation mass spectrometry (ESI MS). Lanes of interest were cut into 10 equal sections, cut from the gel and protein in each section was subjected to trypsin digestion and peptides were analysed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (ThermoScientific) connected to an Agilent 1200 series nano LC system as described in (25). The following criteria were applied to select for pacCer-modified cytosolic proteins: (i) no spectral counts in the controls (no pacLipid/±UV, pacCer/-UV) and ≥3 spectral counts in the pacCer photoaffinity-labeled samples (pacCer/+UV) for Exp.…”

“…Moreover, interactions where the lipid has to enter a deep hydrophobic binding pocket within the protein are likely to be missed. In recent years, bifunctional lipid analogs have emerged as promising new tools to circumvent some of these disadvantages, by guest, on May 11, 2018 www.jlr.org Downloaded from enabling a global profiling of lipid-protein interactions in living cells (25)(26)(27)(28). Bifunctional lipids possess a small diazirine group to allow photo-crosslinking with their protein interaction partners and a terminal alkyne or clickable group for functionalization.…”

Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes

“…However, whether tubulin interacts directly with ceramide and what is the function of this potential interaction remained unclear. To test this, we used a bifunctional ceramide analog (pacFACer) that can be UV-cross-linked to ceramide binding proteins and then derivatized by click chemistry with fluorophores or biotin, a novel labeling technique recently developed for sphingolipids (19,(66)(67)(68). In addition, we used ceramide-linked agarose beads to pull down and identify ceramide binding proteins from cell lysates.…”