Protein Lipidation: Methods and Protocols

✍ Scribed by Maurine E. Linder

• Publisher: Springer New York; Humana
• Year: 2019
• Tongue: English
• Leaves: 316
• Series: Methods in Molecular Biology 2009
• Edition: 1st ed.
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Front Matter ....Pages i-xiv
Front Matter ....Pages 1-1
Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods (Charlotte H. Hurst, Dionne Turnbull, Piers A. Hemsley)....Pages 3-11
Detection of Heterogeneous Protein S-Acylation in Cells (Jennifer Greaves, Nicholas C. O. Tomkinson)....Pages 13-33
Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes (Mina Ahmadi, Kiall Francis Suazo, Mark D. Distefano)....Pages 35-43
Chemical Proteomic Analysis of S-Fatty Acylated Proteins and Their Modification Sites (Emmanuelle Thinon, Howard C. Hang)....Pages 45-57
Direct Analysis of Protein S-Acylation by Mass Spectrometry (Yuhuan Ji, Cheng Lin)....Pages 59-70
Enrichment of S-Palmitoylated Proteins for Mass Spectrometry Analysis (Melanie Cheung See Kit, Brent R. Martin)....Pages 71-79
Front Matter ....Pages 81-81
Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay (Takashi Kanadome, Norihiko Yokoi, Yuko Fukata, Masaki Fukata)....Pages 83-98
Measuring S-Depalmitoylation Activity In Vitro and In Live Cells with Fluorescent Probes (Rahul S. Kathayat, Bryan C. Dickinson)....Pages 99-109
Dynamic Radiolabeling of S-Palmitoylated Proteins (Laurence Abrami, Robin A. Denhardt-Eriksson, Vassily Hatzimanikatis, F. Gisou van der Goot)....Pages 111-127
Fluorogenic Assays for the Defatty-Acylase Activity of Sirtuins (Jun Young Hong, Ji Cao, Hening Lin)....Pages 129-136
Global Profiling of Sirtuin Deacylase Substrates Using a Chemical Proteomic Strategy and Validation by Fluorescent Labeling (Shuai Zhang, Nicole A. Spiegelman, Hening Lin)....Pages 137-147
Front Matter ....Pages 149-149
siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR (Heather McClafferty, Michael J. Shipston)....Pages 151-168
In Vitro Assays to Monitor the Enzymatic Activities of zDHHC Protein Acyltransferases (David A. Mitchell, Laura C. Pendleton, Robert J. Deschenes)....Pages 169-177
Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System (Martin Ian P. Malgapo, Maurine E. Linder)....Pages 179-189
Bioinformatic Identification of Functionally and Structurally Relevant Residues and Motifs in Protein S-Acyltransferases (Rodrigo Quiroga, Javier Valdez Taubas)....Pages 191-199
Front Matter ....Pages 201-201
SwissPalm 2: Protein S-Palmitoylation Database (Mathieu Blanc, Fabrice P. A. David, F. Gisou van der Goot)....Pages 203-214
Front Matter ....Pages 215-215
Probing Interaction of Lipid-Modified Wnt Protein and Its Receptors by ELISA (Aaron H. Nile, Rami N. Hannoush)....Pages 217-225
Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase (Michelle A. Sieburg, Elizabeth R. Cleverdon, James L. Hougland)....Pages 227-241
In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities (James John Asciolla, Kalpana Rajanala, Marilyn D. Resh)....Pages 243-255
Front Matter ....Pages 257-257
Production of Farnesylated and Methylated Proteins in an Engineered Insect Cell System (William Gillette, Peter Frank, Shelley Perkins, Matthew Drew, Carissa Grose, Dominic Esposito)....Pages 259-277
A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24 (Erh-Ting Hsu, Jeffrey S. Vervacke, Mark D. Distefano, Christine A. Hrycyna)....Pages 279-293
Front Matter ....Pages 295-295
Monitoring RhoGDI Extraction of Lipid-Modified Rho GTPases from Membranes Using Click Chemistry (Akiyuki Nishimura, Maurine E. Linder)....Pages 297-306
Purification of the Rhodopsin–Transducin Complex for Structural Studies (Yang Gao, Jon W. Erickson, Richard A. Cerione, Sekar Ramachandran)....Pages 307-315
Reconstitution of the Rhodopsin–Transducin Complex into Lipid Nanodiscs (Yang Gao, Jon W. Erickson, Richard A. Cerione, Sekar Ramachandran)....Pages 317-324
Back Matter ....Pages 325-327

✦ Subjects

Life Sciences; Protein Science; Lipidology; Mass Spectrometry