The aim of the project was to bring together two fields - the field of nutrient sensing and the field of protein post-translational modifications - to uncover a mechanism how nutrients can regulate cell signaling and animal physiology. Prior to the ERC project, we discovered that one protein, the Transferrin Receptor (TfR1) can be differentially post-translationally modified by two different lipids - either palmitic acid (C16:0) or stearic acid (C18:0) - and that the identity of the fatty acid modifying TfR1 affects the ability of TfR1 to signal into the cell. The aim of the ERC project was to generalize this single observation and to see whether differential acylation of proteins with different lipids is a broadly-applicable mechanism for how lipids affect cell signaling.
Protein palmitoylation has been known since several decades as a protein post-translational mechanism whereby palmitic acid is attached to cysteine residues of proteins. More recent mass spectrometry data have revealed that also other fatty acids can be attached to proteins, and indeed in half of all cases, the lipid modifying a protein is a fatty acid other than palmitic acid. It was unclear, however, whether different proteins get acylated with different fatty acids, or whether individual protein species can be acylated with different fatty acids. So the first goal of the ERC project was to understand whether differential acylation of single proteins is a general phenomenon. We employed a proteomics approach to identify the human proteins that are palmitoylated or 'stearoylated' (ie modified with C18:0) and discovered that all proteins that are stearoylated can also be palmitoylated in vivo. We went on to show that in all cases that we tested, the stearoylation or palmitoylation occurs on the same cysteine residues of the protein, meaning that there is competition in vivo for these two modifications. Finally, we showed that exposure of cells to either C16:0 or C18:0 will shift the balance of protein palmitoylation or stearoylation, meaning that the metabolic environment of a cell will influence the post-translational modifications of proteins in the cell. This work was published in the Journal of Biological Chemistry (Nuskova et al. 2023). Surprisingly, this mechanism works not only in cell culture but also in vivo. We found that when people ingest stearic acid, this has differential effects on signaling compared to ingestion of palmitic acid (Senyilmaz-Tiebe Nature Communications 2018). We also published a review describing this novel concept of nutrient sensing whereby metabolite levels affect the stoichiometry of post-translational modifications on proteins in Developmental Cell (Figlia et al. 2020).
The second goal of the ERC project was to identify cases where differential acylation of proteins affects their function, and to understand how. We tested a number of proteins. In some cases, we did not find a differential function for stearoylated versus palmitoylation. For instance, we found that a component of the mTORC1 signaling pathway, LAMTOR1, is palmitoylated or stearoylated, however this did not affect mTORC1 localization or signaling (Task 2.2). In some cases, however, we found a clear effect. For instance, we discovered that the GNAI proteins, which mediate signaling downstream of receptor tyrosine kinases such as EGFR, are either stearoylated or palmitoylated on Cys 3. If they are palmitoylated, they enter specific subdomains of the plasma membrane called detergent-resistant-membranes (DRMs), thereby entering into proximity with EGFR and potentiating oncogenic signaling downstream of EGFR. Instead, if they are stearoylated on Cys 3, surprisingly, we found that this lipid becomes desaturated to C18:1, causing the GNAI proteins to exit DRMs, dampening EGFR signaling. Thus, this work which was published in Nature Communications (Nuskova et al. 2021) not only showed that differential acylation of a protein with different lipids can have differential effects on its activity, but it also revealed the detailed molecular mechanism how this works.
Finally, the goal of the third work package was to study how stearoylation is added and removed from proteins. We identified several different ZDHHC acyl-transferases as being responsible for stearoylating TfR1 and GNAI proteins. What was interesting about this finding was that multiple different acyltransferases can transfer either C16:0 or C18:0 onto proteins, meaning that they are quite promiscuous. Instead, the degree of acylation of proteins with C16:0 versus C18:0 depends on the relative ratio of these two metabolites in cells (Nuskova et al. JBC 2023). Hence, this is a mechanism for metabolite sensing. Finally, regarding removal of stearoylation, we found that this is catalyzed by acyl protein thioesterase 1 (APT1), as it can be inhibited with palmostatin B.
In sum, the objectives of this project were achieved.
