Ubiquitin-dependent protein degradation / edited by Mark Hochstrasser.

Format:

Book

Contributor:

Hochstrasser, Mark W., 1959- editor.

Series:

Methods in enzymology ; Volume 619.

Methods in Enzymology ; Volume 619

Language:

English

Subjects (All):

Ubiquitin.

Physical Description:

1 online resource (386 pages) : illustrations.

Place of Publication:

Cambridge, MA : Academic Press, 2019.

Summary:

Ubiquitination and Protein Stability - Part B, Volume 619, the latest release in the Methods in Enzymology series, highlights new advances in the field, with this updated volume presenting interesting chapters written by an international board of authors. Topics of note include chapters on Assays of SUMO protease function in mammalian cells, In vitro analysis of proteasome-associated USP14 activity for substrate degradation and deubiquitylation, Methods to study proteasome regulatory particle assembly, Native mass spectrometry approaches to study the proteasome, Single-molecule methods to study the ubiquitin-proteasome system, Assays for the function of ubiquitin in the mammalian endocytic pathway, 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

Contents:

Front Cover

Ubiquitin-dependent Protein Degradation

Copyright

Contents

Contributors

Preface

References

Chapter One: Assays for protein retrotranslocation in ERAD

1. Introduction

2. An in vitro assay for retrotranslocation of Hmg2-GFP

2.1. Materials

2.1.1. Strains

2.1.2. Microsome preparation

2.1.3. Cytosol preparation

2.2. In vitro ubiquitination of Hmg2-GFP

2.3. In vitro retrotranslocation assay of Hmg2-GFP

3. An in vivo assay for retrotranslocation of Hmg2-GFP

3.1. Materials

3.1.1. Strains

3.2. Assay of in vivo ubiquitination of Hmg2-GFP

3.3. In vivo retrotranslocation assay of Hmg2-GFP

4. Characterizing the nature of retrotranslocated Hmg2-GFP

4.1. Ubiquitin cleavage from retrotranslocated Hmg2-GFP

4.2. Sucrose gradient flotation assay of retrotranslocated Hmg2-GFP

4.3. Proteomic analysis of retrotranslocated Hmg2-GFP and associated retrochaperones

Acknowledgments

Chapter Two: Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae

2. Expression and isolation of misfolded proteins in yeast

2.1. Choice of expression system

2.2. Choice of misfolded proteins

2.3. Choice of lysis and solubilization methods

2.3.1. Boiling in SDS sample buffer

2.3.2. Bead beating

2.3.3. Cryogrinding

2.3.4. Enzymatic digestion with Zymolyase

3. Assays of protein clearance

3.1. Clearance of misfolded protein puncta

3.2. Cycloheximide chase assay

4. Analysis of ubiquitin linkages

4.1. Immunoprecipitation of ubiquitinated misfolded proteins

4.2. Ubiquitin linkage ELISA

Chapter Three: Methodologies to monitor protein turnover at the inner nuclear membrane

2. Tagging strategy for INM proteins.

2.1. Construct design and stable cell line generation

2.1.1. Cloning

2.1.1.1. Notes

2.1.2. Virus transduction and single cell selection

2.1.2.1. Buffers and reagents

2.1.2.2. Equipment

2.1.2.3. Procedure

2.1.2.4. Notes

2.2. Immunofluorescence analysis of tagged INM proteins

2.2.1. Buffers and reagents

2.2.2. Equipment

2.2.3. Procedure

2.2.3.1. Notes

2.3. Pulse-chase experiment for protein turnover analysis

2.3.1. Buffers and reagents

2.3.2. Equipment

2.3.3. Procedure

2.3.4. SDS-PAGE and autoradiography

2.3.4.1. Notes

3. Fractionation of nuclei into NE and nucleoplasm

3.1. Buffers and reagents

3.1.1. Notes

3.2. Equipment

3.3. Procedure

3.3.1. Notes

4. Concluding remarks

Chapter Four: Assays for dissecting the in vitro enzymatic activity of yeast Ubc7

2. Protein production and purification

2.1. General considerations for recombinant protein production and purification

2.1.1. Bacteria used

2.1.2. Bacterial growth media and reagents

2.1.3. Recombinant protein induction

2.1.4. Cell extraction

2.1.5. Notes

2.2. Production and purification of Ub

2.2.1. Equipment

2.2.2. Reagents and buffers

2.2.4. Notes

2.3. Purification of E1 Ub-activating enzyme

2.3.1. Preparation of Ub-affinity resin

2.3.1.1. Equipment

2.3.1.2. Buffers and reagents

2.3.1.3. Procedure

2.3.1.4. Notes

2.3.2. Covalent-affinity purification of E1 enzyme

2.3.2.1. Equipment

2.3.2.2. Reagents and buffers

2.3.2.3. Procedure

2.3.2.4. Notes

2.4. Purification of Ubc7 and Ubc7: Cue1 heterodimer

2.4.1. Equipment

2.4.2. Reagents and buffers

2.4.3. Procedure

2.5. Purification of Doa10 and Hrd1 RING domains

2.5.1. Materials

2.5.2. Reagents and buffers

2.5.3. Procedure.

2.5.4. Notes

2.6. Synthesis of fluorescent Ub (Ub)

2.6.1. Equipment

2.6.2. Buffers and reagents

2.6.3. Procedure

3. Measurement of Ubc7 enzymatic activity

3.1. Measurement of Ubc7 poly-ubiquitylation activity

3.1.1. Reagents and buffers

3.1.2. Procedure

3.1.3. Notes

3.2. Measurement of Ubc7Ub thioester formation (Ubc7 charging)

3.2.1. Reagents and buffers

3.2.2. Procedure

3.2.3. Notes

3.3. Measurement of a single ubiquitin transfer reaction

3.3.1. Reagents and buffers

3.3.2. Procedure

3.3.3. Notes

4. Final remarks

Acknowledgment

Chapter Five: Methods for genetic analysis of mammalian ER-associated degradation

2. Materials

2.1. Reagents

2.2. Equipment

2.3. Solutions

2.4. Software

3. Methods

3.1. Considerations for screen design

3.1.1. Cell type

3.1.2. Dynamic range

3.1.3. Cas9 variant and sgRNA libraries

3.2. Inducible ERAD reporter cell line generation

3.2.1. Lentiviral preparation and transduction

3.2.2. Cell line validation

3.2.2.1. Optimization of reporter induction

3.2.2.2. Analysis of reporter solubility by sequential detergent extraction of protein lysates

3.2.2.3. Analysis of reporter half-life and UPS dependence by translation shutoff

3.2.2.3.1. Suspension cell lines

3.2.2.3.2. Adherent cell lines

3.2.2.4. Optimization of reporter turnover readout by transcription shutoff

3.2.3. Conducting a screen

3.3. Outlook

4. Notes

Chapter Six: A dual system to manipulate protein levels for DNA replication- and cell cycle-related studies

1.1. Principles of the auxin-inducible degron system

1.2. Principles of Tet-regulated gene expression.

2. Application: Analysis of cross talk between DNA damage bypass and checkpoint signaling in the cell cycle

3. Experimental procedures and materials

3.1. Construction of yeast strains

3.2. Cell cycle assays and analysis of cell survival

3.2.1. Materials and equipment

3.2.2. Scheme A: Assessing the effects of checkpoint signaling on damage bypass during S phase

3.2.3. Scheme B: Assessing the effects of checkpoint signaling on damage bypass after completion of S phase

3.2.4. Scheme C: Assessing the effects of checkpoint reactivation during S phase on damage bypass

3.2.5. Notes

3.3. Preparation and Western blot analysis of total cell extracts

3.3.1. Materials and equipment

3.4. Analysis of cell cycle profiles by flow cytometry

3.4.1. Materials and equipment

3.4.2. Procedure

4. Limitations of the dual expression-control method

5. Outlook

Chapter Seven: Engineered disulfide crosslinking to measure conformational changes in the 26S proteasome

2. Assessing the impact of ATP analogs on 26S proteasome conformational distribution

2.3. Procedure

2.3.1. Cell growth, spheroplasting, and lysis

2.3.2. Crosslinking of WCE

2.3.3. Nonreducing SDS-PAGE immunoblotting and quantitation of crosslinking

3. Assessing the impact of proteasomal peptidase inhibitor bortezomib on 26S proteasome conformational distribution

3.3.1. Disulfide crosslinking of vehicle- or bortezomib-treated WCE

3.3.2. Confirming proteasome inhibition via fluorogenic substrate cleavage assay

4. Summary and conclusion

5. Notes

Chapter Eight: Assays for ubiquitin-like protein ligation and proteasome function in archaea.

1. Introduction

2. Methods for assay of proteasome function in archaea

2.1. Monitoring the levels of proteasome targets in archaea

2.2. Generation of proteasome mutant strains

2.3. Assay of protein stability in proteasome mutant strains

2.4. Immunoblotting analysis and quantification of Ubl-proteasome system targets

3. Methods for reconstitution of archaeal ubiquitin-like protein ligation

3.1. Expression and purification of Ubl ligation system components

3.1.1. E1-like enzyme UbaA purification

3.1.2. Ubl SAMP2 purification

3.1.3. MsrA purification

3.1.4. JAMM1 metalloprotease purification

3.1.5. Cell lysate preparation

3.2. Reconstitution of Ubl-protein ligation

3.3. Cleavage of the Ubl modification

4. Conclusions

Chapter Nine: Structural mass spectrometry approaches to study the 20S proteasome

2. 20S proteasome purification protocols for native MS analysis

2.1. Purification of 20S proteasomes from rat livers

2.1.1. Special equipment

2.1.2. Buffers and reagents

2.1.3. General notes prior to starting the purification process

2.1.4. Details of purification procedure

2.2. Purification of the 20S proteasome from S. cerevisiae

2.2.1. Special equipment

2.2.2. Buffers and reagents

2.2.3. General notes prior to starting the purification process

2.2.4. Procedure

2.3. Purification of the 20S proteasome from HEK293 cells

2.3.1. Special equipment

2.3.2. Buffers and reagents

2.3.3. General notes prior to starting the purification process

2.3.4. Procedure

3. Native mass spectrometry approaches to characterize the 20S proteasome

3.1. General notes

3.2. MS analysis of the intact 20S proteasome

3.2.1. Orbitrap instrumental settings for MS analysis

3.2.2. QTOF instrumental settings for MS analysis.

3.3. MS analysis of the intact 20S proteasome.

Notes:

Includes bibliographical references.

Description based on print version record.

ISBN:

0-12-818668-2

0-12-818667-4