The action “QuantPalm_Immunity”, was implemented at the partner organization, The Rockefeller University (New York, USA) for 2 years, in a lab specialized in lipid modifications of proteins and for one year at the host organization, the Crick (London, UK) in a lab specialized in autophagy. The first objective of the action was to find which proteins involved in the fight to pathogen infection (immune proteins) are modified with palmitic acid, and to study if levels of this lipid modification changes upon infection. Palmitic acid is typically added to Cysteine residues of proteins. The first objective could be accomplished with the development of a method allowing to 1) detect which proteins are modified by the lipid, 2) detect the protein and Cysteine modified and 3) quantify the amount of protein modified by the lipid. This could be done by “mass-spectrometry-based quantitative proteomic”. This analysis method allows to identify which peptides are found in the sample by detecting their mass, which protein they belong to and in what relative quantity they were found (relative quantity compared to other proteins or other samples).
The development of a mass-spectrometry (MS) based quantitative proteomic method proved to be highly challenging due to low solubility of the proteins modified with palmitic acid. We thus developed a proteomic method for the identification of the lipidated proteins and sites. Extensive optimizations were performed to increase the peptide solubility and recovery for MS analysis. Our increased knowledge of peptide solubility will be used in the future to develop a quantitative method to quantify sites modified with palmitic acid. The new method was successfully applied to detect known and novel sites modified with palmitic acid. Detection of modified sites is essential to 1) confirm that the protein is modified by palmitic acid and 2) identify which Cysteines is/are modified, as it could be time consuming to identify the modified Cysteine by more traditional methods.
To study if levels of this lipid modification change upon infection, we used MS based proteomics again and compared the amount of palmitic acid modified proteins in naïve and inflammatory stimulated immune cells (RAW264.7). These changes might reflect changes in protein expression or abundance that often occur following immune stimulation and might not reflect an increase of palmitic acid modification on one specific protein.
Our screen allowed us to map proteins modified with palmitic acid in two different cell lines, RAW264.7 and HeLa cells. We also identified a protein involved in autophagy-mediated clearance of pathogen which was found to be modified with palmitic acid. While its enrichment did not change between stimulated and non-stimulated cells, we hypothesized that this protein might play a key role in host-defense to pathogen infection. These results have recently been published (open access).
The second objective of the work was to study the role of the lipid modification on the protein localization and function. We first confirmed that the protein was indeed modified by palmitic acid and we confirmed the modification site. Levels of palmitic acid on the protein were found unchanged following different stimulus (immune stimulation with IFN-alpha or autophagy activation in HeLa cells). Localization and function of the protein did not change when palmitic acid was removed from the protein. The role of palmitic acid is sometime challenging to study and to understand. However, unpublished data suggests that palmitic acid plays an important role in protein trafficking. The role of palmitic acid modification was further studied at the host institution in the context of Mycobacterium tuberculosis (MTb). We hypothesize that palmitic acid is key for the recruitment of the protein to vesicles containing MTb and this could be an important mechanism for the autophagy-mediated clearance of pathogens.
