Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation

Cells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN-dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, and the HIV-1 matrix protein at its myristoylation site. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.

Protein Acylation by Saturated Very Long Chain Fatty Acids and Endocytosis Are Involved in Necroptosis

<p>Necroptosis is a form of regulated cell death that is characterized by membrane permeabilization. This permeabilization is responsible for the inflammatory properties of necroptosis and is critical for disease states involving this process. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that mixed lineage kinase like protein (MLKL) and phosphoMLKL, key proteins for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of the clathrin-mediated endocytosis increases cell viability during necroptosis, likely by removing phosphoMLKL from the plasma membrane. <br></p>

Protein Acylation by Saturated Very Long Chain Fatty Acids and Endocytosis Are Involved in Necroptosis

<p>Necroptosis is a form of regulated cell death that is characterized by membrane permeabilization. This permeabilization is responsible for the inflammatory properties of necroptosis and is critical for disease states involving this process. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that mixed lineage kinase like protein (MLKL) and phosphoMLKL, key proteins for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of the clathrin-mediated endocytosis increases cell viability during necroptosis, likely by removing phosphoMLKL from the plasma membrane. <br></p>

Computational Identification of Lysine Glutarylation Sites Using Positive- Unlabeled Learning

Background: As a new type of protein acylation modification, lysine glutarylation has been found to play a crucial role in metabolic processes and mitochondrial functions. To further explore the biological mechanisms and functions of glutarylation, it is significant to predict the potential glutarylation sites. In the existing glutarylation site predictors, experimentally verified glutarylation sites are treated as positive samples and non-verified lysine sites as the negative samples to train predictors. However, the non-verified lysine sites may contain some glutarylation sites which have not been experimentally identified yet. Methods: In this study, experimentally verified glutarylation sites are treated as the positive samples, whereas the remaining non-verified lysine sites are treated as unlabeled samples. A bioinformatics tool named PUL-GLU was developed to identify glutarylation sites using a positive-unlabeled learning algorithm. Results: Experimental results show that PUL-GLU significantly outperforms the current glutarylation site predictors. Therefore, PUL-GLU can be a powerful tool for accurate identification of protein glutarylation sites. Conclusion: A user-friendly web-server for PUL-GLU is available at http://bioinform.cn/pul_glu/.