ADP-ribosylation and NAD+ Utilizing Enzymes

This volume focuses on mono-ADP-ribosylation and enzymes that use NAD+ including Sirtuins, PARPs, and bacterial and eukaryotic ADP-ribosyltransferases. The chapters in this book are organized into eight parts, and offer detailed descriptions of key protocols used to study topics such as in vitro techniques for ADP-ribosylation substrate identification; biochemical and biophysical assays of PAR-WWE domain interactions; monitoring expression and enzyme activity of ecto-ARTCs; HPLC-based enzymes assays for Sirtuins; and identifying target RNAs of PARPs. 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 thorough, ADP-ribosylation and NAD+ Utilizing Enzymes: Methods and Protocols is a valuable resource for anyone interested in this developing and expanding field.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
Vitamin B3 in Health and Disease: Toward the Second Century of Discovery.- Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently-Tagged Macrodomains.- In Vitro Techniques for ADP-Ribosylated Substrate Identification.- Assessment of Intracellular Auto-Modification Levels of ARTD10 using Mono-ADP-Ribose-Specific Macrodomains 2 and 3 of Murine Artd8.- Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR Binding Analysis.- Assays for NAD-Dependent Reactions and NAD Metabolites.- Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro.- Methods to Study TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP) Mono-ADP-Ribosyltransferase Activity.- Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events.- Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases.- Monitoring Expression andEnzyme Activity of Ecto-ARTCs.- ADP-Ribosyl-Acceptor Hydrolase Activities Catalyzed by the ARH Family of Proteins.- Mono-ADP-Ribosylhydrolase Assays.- Hydrolysis of ADP-Ribosylation by Macrodomains.- HPLC-Based Enzyme Assays for Sirtuins.- Small Molecule Screening Assay for Mono-ADP-Ribosyltransferases.- A Simple, Sensitive, and Generalizable Plate Assay for Screening PARP Inhibitors.- Non-Localized Searching of HCD Data for Fast and Sensitive Identification of ADP-Ribosylated Peptides.- Quantitative Determination of MAR Hydrolase Residue Specificity In Vitro by Tandem Mass Spectrometry.- Detection of ADP-Ribosylating Bacterial Toxins.- Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation.- Monitoring the Sensitivity of T Cell Populations towards NAD+ Released during Cell Preparation.- Identifying Target RNAs of PARPs.- ADPr-Peptide Synthesis.- Identifying Genomic Sites of ADP-Ribosylation Mediated by Specific Nuclear PARP Enzymes Using Click-ChIP.- Methods for Using a Genetically-Encoded Fluorescent Biosensor to Monitor Nuclear NAD+.