[34] Synthesis and use of iodo-fatty acid analogs

Berthiaume, Luc G.; Peseckis, Steven M.; Resh, Marilyn D.

doi:10.1016/0076-6879(95)50090-1

Cited by 52 publications

(75 citation statements)

References 18 publications

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“…This compound can be synthesized by published methods [13,14]; small aliquots are also available by request from this author. Radio-iodination is accomplished by incubation with Na 125 I-Iodide to generate 125 I-IC16 palmitate Iiodohexadecanoic acid) [14]. Use of 125 I-IC16 palmitate for labeling palmitoylated proteins provides two distinct advantages over [ 3 H]-palmitate.…”

Section: Radiolabeling With 125 I-ic16 Palmitatementioning

confidence: 99%

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Resh

2006

Methods

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Covalent attachment of palmitate to proteins is a post-translational modification that exerts diverse effects on protein localization and function. The three key technical approaches required for an investigator to determine the role of palmitoylation of Your Favorite Palmitoylated Protein (YFPP) are methods to: 1) detect YFPP palmitoylation; 2) alter or inhibit palmitoylation of YFPP; 3) determine the functional significance of altered YFPP palmitoylation. Here, I describe experimental methods to address these three issues. Both radioactive (radiolabeling with 3 H palmitate or 125 I-IC16 palmitate) and non-radioactive (chemical labeling and mass spectrometry) methods to detect palmitoylated proteins are presented. Next, techniques to inhibit protein palmitoylation are described. These include site specific mutagenesis, and treatment of cells with inhibitors of protein palmitoylation, including 2-bromopalmitate, cerulenin, and tunicamycin. Alternative methods to replace palmitate with other fatty acids are also presented. Finally, general approaches to determining the effect of altered palmitoylation status on YFPP association with membranes and lipid rafts, as well as signal transduction, are described.

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Section: Radiolabeling With 125 I-ic16 Palmitatementioning

confidence: 99%

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Resh

2006

Methods

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“…Radioiodination of the iodopalmitate [specific activity of Ϸ2 mCi͞mmol (1 Ci ϭ 37 GBq)] was performed as described in ref. 34. Antibodies and specialized and common reagents were from a variety of suppliers (described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site).…”

Section: Methodsmentioning

confidence: 99%

Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events

Vilas

Corvi

Plummer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…Whereas MMSDH was shown to be fatty-acylated, preliminary studies also indicated that bovine liver glutamate dehydrogenase is likely to be fatty-acylated (10). 2 Whether inhibition of pyruvate dehydrogenase and ADP/ATP translocase by palmitoyl-CoA is mediated by covalent fatty acylation has not been investigated.…”

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confidence: 99%

“…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) 1 (10,11). Whereas MMSDH was shown to be fatty-acylated, preliminary studies also indicated that bovine liver glutamate dehydrogenase is likely to be fatty-acylated (10).…”

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confidence: 99%

“…In addition to playing a role in signal transduction, dynamic protein fatty acylation has been postulated to play a potential role in the regulation of amino acid catabolism (10,11). In the well fed state, amino acids originating from digestion of dietary proteins in the gastrointestinal tract are absorbed into the bloodstream and may be used for protein synthesis.…”

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confidence: 99%

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Regulation of Mitochondrial Carbamoyl-phosphate Synthetase 1 Activity by Active Site Fatty Acylation

Corvi¹,

Soltys²,

Berthiaume³

2001

Journal of Biological Chemistry

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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)

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[34] Synthesis and use of iodo-fatty acid analogs

Cited by 52 publications

References 18 publications

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events

Regulation of Mitochondrial Carbamoyl-phosphate Synthetase 1 Activity by Active Site Fatty Acylation

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