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This volume explores techniques used to detect lipids attached to proteins, to analyze the function of lipid modifications, and to characterize the enzymes that add and remove lipids from proteins. The book is organized into seven parts: Part One describes chemically-based strategies to identify substrates for protein lipidation that can be applied to individual proteins or globally using proteomics. Part Two focuses on the enzymes that remove fatty acids from proteins and provides methods to monitor protein biogenesis and palmitate turnover. Part Three addresses biochemical and cellular characterization of DHHC S-acyltransferases, a family of enzymes with 23 members encoded by the human genome. Part Four presents the SwissPalm 2 database and tips on how to use it effectively. Part Five focuses on fatty acylation that occurs in the lumen of the secretory pathway. Parts Six and Seven conclude the book with methods to produce and assay lipid-modified and integral membrane proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and authoritative, Protein Lipidation: Methods and Protocols is a valuable resource for experts in the field and for investigators who encounter protein lipidation through their research on a particular cellular process or favorite protein.
Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods.- Detection of Heterogeneous Protein S-Acylation in Cells.- Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes.- Chemical Proteomic Analysis of S-Fatty Acylated Proteins and their Modification Sites.- Direct Analysis of Protein S-Acylation by Mass Spectrometry.- Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis.- Systematic Screening of Depalmitoylating Enzymes and Evaluation of their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay.- Measuring S-Depalmitoylation Activity In Vitro and In Live Cells with Fluorescent Probes.- Dynamic Radiolabeling of S-Palmitoylated Proteins.- Fluorogenic Assays for the Defatty-Acylase Activity of Sirtuins.- Global Profiling of Sirtuin Deacylase Substrates Using Chemical Proteomic Strategy and Validation by Fluorescent Labeling.- siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR.- In Vitro Assays to Monitor the Enzymatic Activities of zDHHC Protein Acyltransferases.- Purification of Recombinant zDHHC Proteins Using an Insect Cell Expression System.- Bioinformatic Identification of Functionally and Structurally Relevant Residues and Motifs in Protein S-Acyltransferases.- SwissPalm 2: Protein S-Palmitoylation Database.- Probing Interaction of Lipid-Modified Wnt Protein and Its Receptors by ELISA.- Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase.- In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities.- Production of Farnesylated and Methylated Proteins in an Engineered Insect Cell System.- A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24.- Monitoring RhoGDI Extraction of Lipid-Modified Rho GTPases from Membranes using ClickChemistry.- Purification of the Rhodopsin-Transducin Complex for Structural Studies.- Reconstitution of the Rhodopsin-Transducin Complex into Lipid Nanodiscs.
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