Endothelial Cell Palmitoylproteomic Identifies Novel Lipid-Modified Targets and Potential Substrates for Protein Acyl Transferases

• Published in: Circulation research Vol. 110; no. 10; pp. 1336 - 1344
• Main Authors: Marin, Ethan P, Derakhshan, Behrad, Lam, TuKiet T, Davalos, Alberto, Sessa, William C
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD American Heart Association, Inc 11.05.2012; Lippincott Williams & Wilkins
• Subjects: Acetyltransferases - genetics; Acetyltransferases - metabolism; Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Sequence; Animals; Biological and medical sciences; Chlorocebus aethiops; COS Cells; Endothelial Cells - cytology; Endothelial Cells - enzymology; Fundamental and applied biological sciences. Psychology; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Lipoylation - physiology; Molecular Sequence Data; Platelet Endothelial Cell Adhesion Molecule-1 - metabolism; Proteomics - methods; RNA, Small Interfering - genetics; Substrate Specificity; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; Superoxide Dismutase-1; Vertebrates: cardiovascular system; Vertebrata; Endothelial cell; Mammalia; Enzyme; Transferases; Proteomics; Lipids; Circulatory system; Protein; palmitoylation; Endothelium
• ISSN: 0009-7330; 1524-4571; 1524-4571
• DOI: 10.1161/CIRCRESAHA.112.269514

RATIONALE:Protein S-palmitoylation is the posttranslational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well-understood, partly because of technological limits on palmitoylprotein detection. OBJECTIVE:To develop a method using acyl-biotinyl exchange technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in ECs. METHODS AND RESULTS:More than 150 putative palmitoyl proteins were identified in ECs using acyl-biotinyl exchange and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase-1, an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine-6 prevents palmitoylation, leads to reduction in superoxide dismutase-1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for superoxide dismutase-1 palmitoylation. Moreover, we used acyl-biotinyl exchange to search for substrates of particular protein acyl transferases in ECs. We found that palmitoylation of the cell adhesion protein platelet endothelial cell adhesion molecule-1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of platelet endothelial cell adhesion molecule-1 at the cell surface. CONCLUSIONS:Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important posttranslational lipid modification in EC biology.