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The Ubiquitin Proteasome System

Overview of attention for book
Cover of 'The Ubiquitin Proteasome System'

Table of Contents

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    Book Overview
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    Chapter 1 Characterization of RING-Between-RING E3 Ubiquitin Transfer Mechanisms
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    Chapter 2 Single-Turnover RING/U-Box E3-Mediated Lysine Discharge Assays
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    Chapter 3 Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins
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    Chapter 4 Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for Their Ubiquitin Ligase Partners
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    Chapter 5 Competition Assay for Measuring Deubiquitinating Enzyme Substrate Affinity
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    Chapter 6 Enzymatic Assembly of Ubiquitin Chains
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    Chapter 7 Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions
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    Chapter 8 Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries
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    Chapter 9 Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells
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    Chapter 10 A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting
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    Chapter 11 E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins
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    Chapter 12 Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation
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    Chapter 13 Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway
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    Chapter 14 Methods for Preparing Cryo-EM Grids of Large Macromolecular Complexes
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    Chapter 15 Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes
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    Chapter 16 Native Gel Approaches in Studying Proteasome Assembly and Chaperones
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    Chapter 17 Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways
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    Chapter 18 Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis
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    Chapter 19 Measurement of the Multiple Activities of 26S Proteasomes
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    Chapter 20 Exploring the Regulation of Proteasome Function by Subunit Phosphorylation
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    Chapter 21 Scalable In Vitro Proteasome Activity Assay
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    Chapter 22 Exploring the Rampant Expansion of Ubiquitin Proteomics
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    Chapter 23 Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome
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    Chapter 24 Interpreting the Language of Polyubiquitin with Linkage-Specific Antibodies and Mass Spectrometry
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    Chapter 25 Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry
Attention for Chapter 15: Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes
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Chapter title
Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes
Chapter number 15
Book title
The Ubiquitin Proteasome System
Published in
Methods in molecular biology, September 2018
DOI 10.1007/978-1-4939-8706-1_15
Pubmed ID
Book ISBNs
978-1-4939-8705-4, 978-1-4939-8706-1
Authors

Jared A. M. Bard, Andreas Martin, Bard, Jared A. M., Martin, Andreas

Abstract

The 26S proteasome is the major regulated protease in eukaryotes and is responsible for degrading ubiquitinated substrates. It consists of a barrel-shaped 20S core peptidase and one or two 19S regulatory particles, which recognize, unfold, and translocate substrates into the core. The regulatory particle can be further divided into two multi-subunit complexes: the base and the lid. Here we present protocols for expressing the Saccharomyces cerevisiae base and lid recombinantly in Escherichia coli and purifying the assembled subcomplexes using a tandem affinity purification method. The purified complexes can then be reconstituted with 20S core to form fully functional proteasomes. Furthermore, we describe a method for incorporating the unnatural amino acid p-azido-L-phenylalanine into the recombinant complexes at any residue position, allowing for non-disruptive site-specific modifications of these large assemblies. The use of recombinant proteins allows for complete mutational control over the proteasome regulatory particle, enabling detailed studies of the mechanism by which the proteasome processes its substrates. The ability to then specifically modify residues in the regulatory particle opens the door to a wide range of previously impossible biochemical and biophysical studies. The techniques described below for incorporating unnatural amino acids into the proteasomal subcomplexes should be widely transferable to other recombinant proteins, whether individually purified or in larger multi-subunit assemblies.

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Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 44 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 44 100%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 16 36%
Student > Master 5 11%
Student > Doctoral Student 5 11%
Researcher 3 7%
Professor > Associate Professor 2 5%
Other 5 11%
Unknown 8 18%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 23 52%
Agricultural and Biological Sciences 6 14%
Physics and Astronomy 2 5%
Chemistry 2 5%
Psychology 1 2%
Other 2 5%
Unknown 8 18%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 September 2018.
All research outputs
#15,409,382
of 22,919,505 outputs
Outputs from Methods in molecular biology
#5,360
of 13,132 outputs
Outputs of similar age
#213,788
of 340,023 outputs
Outputs of similar age from Methods in molecular biology
#91
of 235 outputs
Altmetric has tracked 22,919,505 research outputs across all sources so far. This one is in the 22nd percentile – i.e., 22% of other outputs scored the same or lower than it.
So far Altmetric has tracked 13,132 research outputs from this source. They receive a mean Attention Score of 3.4. This one is in the 44th percentile – i.e., 44% of its peers scored the same or lower than it.
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