Non-Lysine Acyl Modifications and Their Effects on Cellular Function

In recent years, our understanding of the scope and diversity of protein post-translational modifications has rapidly expanded. While mitochondrial proteins in particular have been studied in detail and are decorated with an array of acyl groups that can occur non-enzymatically, cytosolic proteins are also known to be acylated. Interestingly, these modifying chemical moieties are often associated with intermediary metabolites from core metabolic pathways. We looked to explore the emerging links between the intrinsic reactivity of metabolites, non-enzymatic protein acylation, and possible signaling roles for these metabolites. Previously HMG-CoA was identified as a reactive metabolite that non-enzymatically modifies proteins when its utilization is blocked. We investigated whether statins, which inhibit the HMG-CoA consuming enzyme HMG-CoA reductase (HMGCR), would result in HMGylation as well. This is particularly relevant because statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Using mass spectrometry based metabolomics, we found that in both cells and mice treated with statins, HMG-CoA levels increased. Surprisingly, this increase in HMG-CoA led to a single protein being HMGylated, which is in contrast to prior work showing a multitude of protein modifications occurring when acyl-CoA consuming enzymes were altered. Using mass spectrometry, we identified the modified protein as fatty acid synthase (FAS), which implied a new connection between two lipid biosynthetic pathways. We thoroughly characterized the chemical nature of the modification through in vitro chemical treatments. These investigations revealed the modification on FAS to be labile and dynamic compared to previously described acyl modifications. The modification was susceptible to heating, reducing agents, as well as displacement by other acyl-CoAs. Additionally, the modification was rapidly induced and removed in cells in response to the addition or removal of statin. This pointed to a modification that was not lysine bound, which is the commonly reported acyl modification, and is very stable. We developed mass spectrometry methods that would stabilize the modification on FAS and were able to locate the site of HMGylation. The modification occurs on both the serine residue responsible for accepting acyl groups, as well as on the prosthetic group that facilitates the growing of the fatty acyl chain. These modifications occur on active site residues and when tested using purified HMGylated FAS, we found the activity to be inhibited in proportion with the amount of HMG-CoA present. We then tested the impact of statin-treatment on the production of fatty acids in both cells and mice. Surprisingly, we found no change in the lipogenesis rates during statin-treatment, despite the in vitro evidence indicating FAS is inhibited when HMGylated. Recent discoveries have shown a role for FAS outside of the canonical production of lipids for energy storage. With the knowledge that there are subpools of FAS localized within the cell, some of which perform signaling functions, we predicted that the HMGylation occurred on a subpool of FAS in close proximity to HMGCR and endoplasmic reticulum (ER) membrane and conveyed a signal. Utilizing cell fluorescence microscopy, we confirmed the interaction of FAS and HMGCR. To look for any possible changes in cellular signaling we utilized label-free quantitative proteomics and identified pathways changing due to the HMGylation of FAS. While the specific mechanism and nature of these changes remains to be discovered, we identified several signatures of ER and Golgi pathways changing in response to FAS HMGylation and discuss possible areas of interest for future investigation. Finally, we used this new information to begin an investigation into broader non-lysine linked modifications. By artificially inducing acylation in cells, we show that heat or reducing agents play a large role in the detection of acyl-PTMs by Western blotting across HMGylation, glutarylation, and acetylation. In summary, we show how the inhibition of a single enzyme can result in the targeted modification of a protein in an adjacent pathway. This modification occurs on a serine residue and prosthetic group, allowing for dynamic modification and regulation of the enzyme. Furthermore, the discovery of these non-lysine residues reveals the possibility for widespread non-lysine acyl PTMs, waiting to be discovered following the revision of current standard methodology.