SUMO Protocols

The functions of the ubiquitin-related modifier SUMO are highly diverse and largely independent of the ubiquitin system. In this volume, leading experts present methods that highlight SUMO's unique biological features in order to support future SUMO research.

Post-translational protein modifications by members of the ubiquitin family are widely recognized as important regulatory control systems for a variety of biological pathways. Their influence on eukaryotic cellular metabolism is comparable to that of other modifi- tions such as phosphorylation, acetylation and methylation. The small ubiquitin-related modifier SUMO uses a conjugation and de-conjugation system closely related to that of ubiquitin itself; yet, the functions of the SUMO system are highly diverse and largely independent of the ubiquitin system. SUMO modification controls the activity of tr- scription factors and can influence protein stability, but it also contributes to nucleo-cy- plasmic transport, chromosome segregation and DNA damage repair. As a consequence, the SUMO system pervades virtually all areas of basic molecular and cell biology, and scientists from different backgrounds, including medical researchers, are likely to enco- ter SUMO in the course of their studies. This volume, SUMO Protocols, therefore aims at presenting a collection of methods relevant to SUMO research in order to make these tools available to biochemists, molecular and cell biologists as well as research-oriented clinicians not yet familiar with the system. In contrast to the ubiquitin system, which has been the subject of several reviews and methods collections, no practical compendium entirely devoted to SUMO has been p- lished.

First methods collection devoted entirely to the SUMO system Provides comprehensive practical advice from leading experts in the field Covers a range of complementary approaches for SUMO target identification Presents step-by-step protocols for the purification and manipulation of SUMO conjugation and de-conjugation enzymes Includes a description of SUMO fusion technology for improved recombinant protein production Includes supplementary material: sn.pub/extras

Klappentext

While the small ubiquitin-related modifier SUMO uses a conjugation and de-conjugation system closely related to that of ubiquitin itself, its functions are highly diverse and largely independent of the ubiquitin system. In SUMO Protocols, leading experts present a collection of methods intended to highlight important features unique to the biology of SUMO in order to support future SUMO research. The first half of the volume focuses on the identification and functional characterization of SUMO targets and interactors, while the second half covers essential biochemical methods for the manipulation of the enzymatic components of the SUMO system. Written in the highly successful Methods in Molecular Biology™ series format, the chapters include brief introductions to the subject, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and the Notes sections, which provide tips on troubleshooting and avoiding known pitfalls.

Unique and cutting-edge, SUMO Protocols provides the necessary tools for biochemists, molecular and cellular biologists as well as research-oriented clinicians not yet familiar with this vital system.

Inhalt
The SUMO System: An Overview.- Identification of Sumo Targets.- Identification of SUMO Target Proteins by Quantitative Proteomics.- Identification of SUMO-Conjugated Proteins and their SUMO Attachment Sites Using Proteomic Mass Spectrometry.- Identification of SUMO Targets Through In Vitro Expression Cloning.- Enhanced Detection of In Vivo SUMO Conjugation by Ubc9 Fusion-Dependent Sumoylation (UFDS).- In Vivo Detection and Characterization of Sumoylation Targets in Saccharomyces cerevisiae.- Identification Of Sumo Interactors.- Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis.- Identification of SUMO-Binding Motifs by NMR.- Functional Analysis Of Sumoylation In Vivo.- Regulation of Transcription Factor Activity by SUMO Modification.- Characterization of the Effects and Functions of Sumoylation Through Rapamycin-Mediated Heterodimerization.- Biochemical Studies Of Sumo Conjugation.- Purification of SUMO Conjugating Enzymes and Kinetic Analysis of Substrate Conjugation.- Performing In Vitro Sumoylation Reactions Using Recombinant Enzymes.- Preparation of Sumoylated Substrates for Biochemical Analysis.- Strategies for the Expression of SUMO-Modified Target Proteins in Escherichia coli.- Biochemical Studies of Sumo Deconjugation.- Preparation of SUMO Proteases and Kinetic Analysis Using Endogenous Substrates.- An In Vitro FRET-Based Assay for the Analysis of SUMO Conjugation and Isopeptidase Cleavage.- FRET-Based In Vitro Assays for the Analysis of SUMO Protease Activities.- Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay.- Manipulation And Alternative Uses Of The Sumo System.- Inhibition of the SUMO Pathway by Gam1.- SUMO Fusion Technology for Enhanced Protein Production in Prokaryotic and Eukaryotic Expression Systems.