Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

p21-activated protein kinase (PAK) 2 is a small GTPase-activated serine͞threonine kinase regulating various cytoskeletal functions and is cleaved by caspase-3 during apoptosis. We demonstrate that the caspase-cleaved PAK2 C-terminal kinase fragment (C-t-PAK2) is posttranslationally myristoylated, although myristoylation is typically a cotranslational process. Myristoylation and an adjacent polybasic domain of C-t-PAK2 are sufficient to redirect EGFP from the cytosol to membrane ruffles and internal membranes. Membrane localization and the ability of C-t-PAK2 to induce cell death are significantly reduced when myristoylation is abolished. In addition, the proper myristoylation-dependent membrane localization of C-t-PAK2 significantly increased signaling through the stress-activated c-Jun N-terminal kinase signaling pathway, which often regulates apoptosis. Interestingly, C-t-PAK2 promoted cell death without compromising mitochondrial integrity. Posttranslational myristoylation of caspase-cleaved proteins involved in cytoskeletal dynamics (e.g., PAK2, actin, and gelsolin) might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis.apoptosis ͉ cytoskeleton ͉ mitochondria ͉ membrane ͉ acylation

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time-and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acylCoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.The covalent modification of proteins by lipids alters their physical and functional properties. Several types of lipids are covalently bound to proteins as follows: isoprenoids, glycosylphosphatidylinositols, cholesterol, and fatty acids (1-4). Protein fatty acylation is the modification of proteins by fatty acids. It is divided into two categories, myristoylation and palmitoylation. In myristoylation, the 14-carbon myristate is co-translationally attached to the N-terminal glycine residue of a protein via a stable amide bond. Palmitoylation is characterized by the post-translational attachment of the 16-carbon fatty acid palmitate to cysteine residues of a protein via a thioester bond. Due to its reversible nature, palmitoylation has been shown recently to regulate the subcellular localization of several proteins involved in signal transduction processes (1,5,6). For protein palmitoylation to occur, palmitate needs to be activated in the form of its coenzyme A derivative, palmitoyl-CoA.Interestingly, palmitoyl-CoA, the acyl donor for protein palmitoylation, inhibits several enzymes including rat adipocyte pyruvate dehydrogenase (7), rat liver ADP/ATP translocase (8), bovine liver glutamate dehydrogenase (9), and bovine liver methylmalonyl semialdehyde dehydrogenase (MMSDH)

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Sitedirected mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and ⅐NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker ␤-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the ⅐NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization. The gaseous radical nitric oxide (⅐NO)1 modulates biological function in a wide range of tissue types, acting either as a signaling molecule or as a toxin. Three human NOS isoforms have been cloned and characterized. Among them, NOS2(sometimes referred to as inducible NOS or iNOS) is mostly involved in the synthesis of the large amounts of ⅐NO that appear in inflammatory and immunologic processes (1, 2).Both crystallographic and enzymatic studies performed with recombinant proteins expressed in Escherichia coli have shown that the N terminus end of the three mammalian NOSs is not involved in ⅐NO synthesis but rather in subcellular targeting of the mature polypeptide chain (2, 3). For instance, the PDZ domain of NOS1 (residues 1-90) interacts with dystrophin and becomes localized to the sarcolemma of fast twitch fibers (4). In fact, deletion of the first 226 amino acids of NOS1 results in a catalytic protein that synthesizes ⅐NO at a similar rate than the full-length protein (5). Likewise, the N-terminal end of NOS3 is covalently and irreversibly myristoylated at Gly-2 and reversibly palmitoylated at Cys-15 and Cys-26 in a well described process responsible for its targeting to caveolae (6, 7). In addition, deletion of the first 52 amino acids of NOS3 does not affect catalytic activity, reflecting that this sequence stretch is not part of the enzymatic machinery but is involved in intrac...

Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In the recent years protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes. Table S1: List of all non-redundant identified candidate proteins from the three independent ABE assays. The first worksheet tab contains a multiconsensus analysis showing proteins identified in all three assays together with their respective mass spectrometry information and in silico predictions. Highlighted in blue are those already described to palmitoylated, in grey are those predicted to be palmitoylated but without experimental evidence and in green those that have homology to known S-acylated proteins from P. falciparum. Tabs 2, 3 and 4 contain particular information and NRP calculations for each assay, while the last tab is a summary of the calculated NRP HA+/HA-ratios calculated for each protein.