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Modifications and targeting of protein termini Part B

1st Edition - July 25, 2023

Editor: Thomas Arnesen

Hardback ISBN:

9 7 8 - 0 - 4 4 3 - 2 2 1 0 0 - 2

eBook ISBN:

9 7 8 - 0 - 4 4 3 - 2 2 1 0 1 - 9

Modifications and Targeting of Protein Termini, Part B, Volume 686 in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting… Read more

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Modifications and Targeting of Protein Termini, Part B, Volume 686 in the Methods in Enzymology serial, highlights new advances in the field with this new volume presenting interesting chapters on a variety of timely topics, including In vitro production of N-degron fused proteins and its application, Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry-based proteomics, Monitoring ADO-dependent proteolysis in cells using fluorescent reporter proteins, Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway, Characterization and chemical modulation of p62/SQSTM1/Sequestosome-1 as an autophagic N-recognin of the Arg/N-degron pathway.

Other chapters cover Analysis of higher plant N-degron pathway components and substrates via expression in S. cerevisiae, Building libraries to dissect terminal degrons with fluorescent timers, Affinity isolation and biochemical characterization of N-degron ligands using the N-recognin, ClpS, Probing the effects of N-terminal acetylation on α-synuclein structure, aggregation and toxicity, Increasing the coverage of the N-terminome with Lys-N Amino Terminal enrichment (LATE), and more.

Cover
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface – The impact of protein N- and C-terminal modifications
Reference
Chapter One: Increasing the coverage of the N-terminome with LysN amino terminal enrichment (LATE)
Abstract
1: Introduction
2: Proteomic sample preparation
3: Data analysis
4: Alternative methods/procedures
References
Chapter Two: Using cell lysates to assess N-terminal acetyltransferase activity and impairment
Abstract
1: Introduction
2: Materials and equipment
3: Method
4: Summary and conclusions
References
Chapter Three: Probing the effects of N-terminal acetylation on α-synuclein structure, aggregation and cytotoxicity
Abstract
1: Introduction
2: Equipment and reagents
3: Protein production
4: Preparation of NTA α-synuclein for aggregation kinetics
5: Kinetic analysis of the aggregation process of NTA α-synuclein
6: Cell toxicity assays
7: Summary and conclusions
Acknowledgments
References
Chapter Four: Identification of N-degrons and N-recognins using peptide pull-downs combined with quantitative mass spectrometry
Abstract
1: Introduction
2: Method overview and work flow
3: General considerations for peptide design
4: Preparation of lysates for peptide pull-downs
5: Binding of biotinylated peptides to streptavidin beads
6: Peptide pull-down assay
7: On beads digest and preparation for MS analysis
8: LC-MS/MS measurement
9: Analysis of mass spectra using MaxQuant
10: Analysis of MaxQuant output files with Perseus
11: Conclusions and outlook
Acknowledgments
References
Chapter Five: In vitro production of N-degron fused proteins and its application
Abstract
1: Introduction
2: Key resources
3: Preparation of N-degron attached ZZ-domain of p62/SQSTM1
4: Preparation of human ATG4B protease
5: Applications of N-degron fused ZZ-p62 or N-degron fused proteins of interest
6: Conclusions
Acknowledgments
References
Chapter Six: TEV protease cleavage in generation of artificial substrate proteins bearing neo-N-termini
Abstract
1: Introduction
2: General method and equipment
3: Construction of a self-cleaving fusion protein
4: Construction of fusion proteins containing the TEV protease recognition site
5: Expression of proteins
6: SDS PAGE and western blot
7: Summary and conclusion
Acknowledgments
References
Chapter Seven: Affinity isolation and biochemical characterization of N-degron ligands using the N-recognin, ClpS
Abstract
1: Introduction
2: Determine N-degron specificity of ClpS (N-recognin) using immobilized peptide arrays
3: ClpS affinity chromatography to isolate (and identify) natural N-degron substrates/ligands
References
Chapter Eight: Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway
Abstract
1: Introduction
2: Preparation of N-degrons for pulldown assays
3: Preparations of N-recognins for pulldown assays
4: N-degron pulldown assays
5: Monitoring the proteome captured by N-degrons
6: Pulldown of N-degron substrates using ubiquitin-fusion technique (UFT)
7: Summary and conclusions
Acknowledgments
References
Chapter Nine: In vitro autoubiquitination activity of E3 ubiquitin ligases of the N-degron pathway
Abstract
1: Introduction
2: General method and equipment
3: Design of constructs
4: Recombinant protein expression of E3 ligase
5: Protein purification after recombinant expression via IMAC
6: In vitro autoubiquitination assay
7: Western blot analyses
Acknowledgments
References
Chapter Ten: Analysis of higher plant N-degron pathway components and substrates via expression in S. cerevisiae
Abstract
1: Introduction
2: Conclusions
Acknowledgments
References
Chapter Eleven: Characterization and chemical modulation of p62/SQSTM1/Sequestosome-1 as an autophagic N-recognin
Abstract
1: Introduction
2: General method and statistical analysis
3: In vitro p62 oligomerization assay
4: Colocalization assay (immunocytochemistry)
5: Autophagic degradation assay
6: Molecular modeling and virtual screening of autophagy targeting ligands
7: Summary and conclusions
Acknowledgments
References
Chapter Twelve: Monitoring ADO dependent proteolysis in cells using fluorescent reporter proteins
Abstract
1: Introduction
2: Design considerations
3: Construction of DNA vector
4: Expression in mammalian cells
5: Immunoblot expression analysis
6: Fixed cell fluorescence analysis
7: High-throughput live cell fluorescence analysis
8: Further considerations
9: Conclusions
References
Chapter Thirteen: Building yeast libraries to dissect terminal degrons with fluorescent timers
Abstract
1: Introduction
2: Design and applications of libraries for MPS profiling
3: Preparation of the reporter plasmid
4: Preparation of DNA oligonucleotides
5: Library construction by homologous recombination in yeast
6: Summary
Acknowledgments
References
Chapter Fourteen: Multiplexed protein stability (MPS) profiling of terminal degrons using fluorescent timer libraries in Saccharomyces cerevisiae
Abstract
1: Introduction
2: Stability based sorting of tFT-libraries
3: Next-generation sequencing (NGS) and stability profiling
4: Conclusion
Acknowledgments
References
Chapter Fifteen: Characterization of degradation signals at protein C-termini
Abstract
1: Introduction
2: General considerations and experimental design
3: Determining the degron potency of C-terminal peptides by GPS assays
4: Unbiased characterization of the features of a C-degron motif using a GPS random peptide library platform
5: Conclusion and perspectives
Acknowledgments
References

Thomas Arnesen

Professor Thomas Arnesen received his Ph.D. in molecular biology from the University of Bergen, Norway in 2006. After postdoctoral work at Haukeland University Hospital and University of Rochester Medical Center, he established his own lab at the University of Bergen in 2010. His main interest has been protein N-terminal acetylation and the responsible enzymes, the N-terminal acetyltransferases (NATs). Using Saccharomyces cerevisiae and human cell models combined with in vitro approaches his lab and collaborators have i) identified and defined the presumed complete cytosolic human NAT-machinery including NATs acting post-translationally, ii) quantitatively analysed the N-terminal acetylomes of yeast and human cells, iii) developed novel assays for NAT-profiling, iv) gained mechanistic insights of the molecular and cellular effects of N-terminal acetylation, v) contributed to the understanding of the physiological and clinical importance of NATs by revealing the links between NatA and cancer cell survival and drug sensitisation, and lately by defining genetic disorders caused by pathogenic NAT variants. The Arnesen lab also contributed to solving the first NAT-structures and developing the first potent NAT-inhibitors. With Fred Sherman and Bogdan Polevoda, Arnesen introduced the NAA (N-alpha acetyltransferase) nomenclature of the N-terminal acetyltransferase genes and proteins, and he acts as the specialist advisor for the HUGO Gene Nomenclature Committee for these genes. Arnesen is one of the founders and council members of the International Society of Protein Termini (ISPT). He has organized several symposia on N-terminal acetylation, and in 2022 he was the head organizer of the EMBO Workshop ‘Protein Termini – From mechanism to biological impact’ in Bergen, Norway. Arnesen has co-authored more than 100 peer-reviewed publications. Today he is head of the Translational Cell Signaling and Metabolism group at the Dept. of Biomedicine, UiB, supported by the Research Council of Norway and ERC. Here his team continues the basic and translational research to understand the impact of protein N-terminal modifications.

Affiliations and expertise

Researcher, Professor, The Department of Biomedicine, Department of Biological Sciences (BIO), Arnesen Lab, University of Bergen, Bergen, Norway