Methods in enzymology. Volume 618, Ubiquitin and ubiquitin-like protein modifiers / edited by Mark Hochstrasser.

Format:

Book

Contributor:

Hochstrasser, Mark W., 1959- editor.

Language:

English

Subjects (All):

Ubiquitin.

Physical Description:

1 online resource (432 pages)

Place of Publication:

Cambridge, Massachusetts : Academic Press, 2019.

Summary:

Ubiquitination and Protein Stability - Part A Volume 618, the latest release in the Methods in Enzymology series, highlights new advances in the field, with this updated volume presenting interesting chapter written by an international board of authors. Topics of note in this new release include the Preparation of ubiquitinated nucleosomes with native and non-hydrolyzable linkages, Methods to measure ubiquitin chain length and linkage, Genetic approaches to study the yeast ubiquitin system, Enzymatic preparation of monoubiquitinated proteins, Methods to distinguish the function of ubiquitin in autophagy and the proteasome pathway, the Purification and characterization of enzyme activity of USPs, and much more.- Provides the authority and expertise of leading contributors from an international board of authors- Presents the latest release in this series on enzymology- Updated release includes the latest information on methods to measure ubiquitin chain length and linkage, genetic approaches to study the yeast ubiquitin system, amongst many other timely topics

Contents:

Front Cover

Ubiquitin and Ubiquitin-like Protein Modifiers

Copyright

Contents

Contributors

Preface

References

Chapter One: Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage

1. Introduction

2. Preparation of dichloroacetone-linked H2B-Ub

2.1. Expression and purification of His-tagged UbG76C

2.1.1. Equipment

2.1.2. Buffers and reagents

2.1.3. Procedure

2.2. DCA linkage reaction

2.2.1. Buffers and reagents

2.2.2. Procedure

2.3. Purification of H2B-UbDCA

2.3.1. Buffers and reagents

2.3.2. Procedure

3. Preparation of native H2B-Ub

3.1. MESNa-mediated cleavage of H2B(1-114)-GyrA-CBD intein to produce H2B-MES

3.1.1. Equipment

3.1.2. Buffers and reagents

3.1.3. Procedure

3.2. Peptide synthesis of Cys(116-122)H2B fragment

3.2.1. Chemical Synthesis of Cys(116-122)H2B peptide fragment

3.2.1.1. Equipment

3.2.1.2. Materials

3.2.1.3. Procedure

3.3. Ub-MES derivatization

3.3.1. Materials

3.3.2. Procedure

3.4. Semisynthesis of native H2BK120Ub

3.4.1. Equipment

3.4.2. Buffers and reagents

3.4.3. Procedure

4. Notes

5. Summary and conclusion

Acknowledgments

Chapter Two: Dual-color pulse-chase ubiquitination assays to simultaneously monitor substrate priming and extension

2. Generate fluorescently labeled ubiquitin (UB)

2.1. Experimental procedure

3. Generating thioester-linked E2UB intermediates for the pulse reaction

3.1. Experimental procedure

4. Test the stability of the E2UB thioester following EDTA addition

4.1. Experimental procedure

5. Pulse-chase assays to monitor a single enzyme activity

5.1. Experimental procedure

6. Dual-color pulse-chase assays monitoring substrate ``priming´´ with UB and extension of polyUB chains

7. Discussion.

References

Chapter Three: A genetic approach to study polyubiquitination in Saccharomyces cerevisiae

2. Strain engineering

2.1. Deletion of ubiquitin sequences from UBI1 and UBI2

2.2. Design of ubiquitin loci encoding ubiquitin variants

2.3. Construction of strains expressing ubiquitin variants

3. Modification of the conventional SGA method to establish an SK1 4-marker ubiquitin SGA protocol

3.1. Drug concentrations

3.2. Sporulation

3.3. Overview of the 4-marker SGA workflow

4. Analysis of data quality

4.1. Calculation and characterization of genetic interaction scores

4.2. Quality determination

5. Identification and characterization of genetic interactions

5.1. Identification of hits specific to individual ubiquitin lysine mutants

5.2. Functional characterization of individual genetic interactions

5.3. Functional analysis of genetic interactomes

6. Validation of genetic interactions

6.1. Mating

6.2. Sporulation

6.3. Tetrad dissection

6.4. Spot dilution assays to validate genetic interactions

7. Single-lysine ubiquitin variants to study ubiquitination in vivo

7.1. Conditional expression of single-lysine ubiquitin variants as the sole ubiquitin source in vivo

8. Concluding remarks

Chapter Four: Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins

2. Cloning of expression constructs

3. Generation of recombinant proteins required for preparing monoubiquitinated FANCD2 and FANCI

3.1. Ube2T (E2) and FANCL (E3) enzymes

3.1.1. Expression and purification of Ube2Tv4

3.1.2. Expression and purification of FANCL

3.2. SpyCatcher and SpyTag ubiquitin proteins

3.3. FANCD2 and FANCI substrates

3.3.1. Bacmid preparation

3.3.2. Generation of baculovirus.

3.3.3. Expression and purification of FANCD2 and FANCI substrates

4. Purification of monoubiquitinated FANCD2 and FANCI

4.1. Preliminary ubiquitination reactions

4.2. Reaction scale-up and purification of monoubiquitinated human FANCD2 and human FANCI

5. Characterization of monoubiquitinated human FANCD2 and human FANCI

5.1. Analytical size-exclusion chromatography of monoubiquitin-conjugated and -unconjugated substrates

5.2. Deubiquitination of ubiquitin-conjugated substrates

6. Concluding remarks

Chapter Five: Methods to measure ubiquitin chain length and linkage

2. Ub-AQUA/PRM analysis to quantify ubiquitin linkages and K48/K63 branched chains

2.1. In-gel digestion and purification of ubiquitin peptides

2.1.4. Notes

2.2. In-gel digestion and purification of branched ubiquitin chain-derived peptides

2.2.3. Notes

2.3. Preparation and quality control of AQUA peptides

2.3.1. Equipment

2.3.2. Buffers and reagents

2.3.3. Procedure

2.3.4. Notes

2.4. PRM for ubiquitin quantification

2.4.1. Equipment

2.4.2. Buffers and reagents

2.4.3. Procedure

2.4.4. Notes

3. Ub-ProT analysis to measure ubiquitin chain length of in vitro and in vivo ubiquitin conjugates

3.1. Purification of TR-TUBE for Ub-ProT analysis

3.1.4. Notes

3.2. Ub-ProT for in vitro synthesized ubiquitin chains

3.2.1. Equipment

3.2.2. Buffers and reagents

3.2.3. Procedure

3.2.4. Notes

3.3. Ub-ProT analysis of ubiquitin conjugates from Saccharomyces cerevisiae

3.3.1. Equipment

3.3.2. Buffers and reagents

3.3.3. Procedure

3.3.4. Notes.

4. Summary and conclusions

Chapter Six: Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE

2. Detection of substrate ubiquitination by a given E3 using TR-TUBE

2.1. Comparison of TR-TUBE and ubiquitin overexpression methods

3. Identification of substrates for a given E3 ligase using TR-TUBE

3.1. Equipment

3.2. Buffers and reagents

3.3. Procedure

3.4. Notes

4. Conclusion

Chapter Seven: Methods to study phosphoribosylated ubiquitin ligation and removal

2. Materials

2.1. L. pneumophila infection

2.2. In vitro ubiquitination assay

3. Methods

3.1. Ubiquitination of Rab33b during L. pneumophila infection

3.1.1. Preparation of L. pneumophila for infection

3.1.2. Preparation of mammalian cells expressing 4xFlag-Rab33b and FCγRII

3.1.3. Bacterial infection and 4xFlag-Rab33b immunoprecipitation

3.1.4. Analyze ubiquitinated Rab33b from cells infected with L. pneumophila

3.2. In vitro ubiquitination of Rab33b by SdeA

3.2.1. Protein purification

3.2.2. Ubiquitination reaction

3.3. Ubiquitination of Rab33b by purified ADP-ribosylated ubiquitin and an SdeA mutant defective in the mART activity

3.3.1. Preparation of activated ubiquitin, ADPR-Ub

3.3.2. Ubiquitination of Rab33b by ADPR-Ub and SdeAE/A, a mutant defective in the mART activity

3.4. In vitro deubiquitination of Rab33b by SidJ

3.4.1. Preparation of Rab33b-Ub

3.4.2. Purification of His6-SidJ from L. pneumophila

3.4.3. Deubiquitination assay

Chapter Eight: Biochemical characterization of SUMO-conjugating enzymes by in vitro sumoylation assays

1. Introduction.

2. Substoichiometric substrate modification

2.1. Equipment

2.2. Buffers and reagents

2.3. Procedure

2.4. Notes

3. E3-mediated E2 discharge

4. E3-E2 backside interaction

4.1. Equipment

4.2. Buffers and reagents

4.3. Procedure

4.4. Notes

5. Donor SUMO positioning

5.1. Equipment

5.2. Buffers and reagents

5.3. Procedure

5.4. Notes

6. Summary and conclusions

Author contributions

Chapter Nine: Methods to study SUMO dynamics in yeast

2. General equipment, yeast strains, and plasmids

2.1. General equipment

2.2. Yeast strains

2.3. Plasmids

3. Western blot analysis of cellular SUMO conjugates to study effects of stress conditions and mutations affecting enzyma ...

3.1. Theory

4. Probing the role of desumoylation, SUMO chain formation, and proteolysis in SUMO dynamics using HA-tagged SUMO variants

4.1. Theory

4.2. Reagents

5. Purification of 8xHis-SUMO conjugates using cobalt beads

5.1. Theory

Chapter Ten: Approaches for investigating the extracellular signaling function of ISG15

2. Methods

2.1. Bacterial expression and purification of ISG15

2.2. Cells and cell culture

2.3. ELISAs for cytokine secretion

2.4. Assays for ISG15-dependent SFK activation

Chapter Eleven: Analysis of modification and proteolytic targeting by the ubiquitin-like modifier FAT10

1.1. The ubiquitin-like modifier FAT10

1.2. The challenge to express soluble, recombinant FAT10 for use in in vitro studies.

1.3. In vitro FAT10ylation assays using recombinant FAT10.

Notes:

Description based on print version record.

ISBN:

0-12-816360-7

0-12-816359-3