Modifications and Targeting of Protein Termini Part A

1st Edition, Volume 684 - May 23, 2023
Editor: Thomas Arnesen
Language: English
Hardback ISBN:
9 7 8 - 0 - 4 4 3 - 1 5 7 7 2 - 1
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 1 5 7 7 3 - 8

Modifications and Targeting of Protein Termini, Volume 684 in the Methods in Enzymology series serial highlights new advances in the field, with this new volume presenting inter… Read more

Modifications and Targeting of Protein Termini Part A

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Modifications and Targeting of Protein Termini, Volume 684 in the Methods in Enzymology series serial highlights new advances in the field, with this new volume presenting interesting chapters pn a variety of timely topics, including Optimizing purification and activity assays of N-terminal methyltransferase complexes, In vitro reconstitution of hierarchical steps of Arg/N-degron pathway, Identification of N-degrons and N-recognins by using peptide-pull-downs combined with quantitative proteomics, A decoupled Virotrap approach to study the interactomes of N-terminal proteoforms, Monitoring ADO-dependent proteolysis in cells using fluorescent reporter proteins, Site-specific α-N-terminal methylation on peptides through chemical and enzymatic methods, and more.

Additional sections cover Monitoring the interactions between N-degrons and N-recognins of the Arg/N-degron pathway, Characterization and chemical modulation of p62/SQSTM1 as an autophagic N-recognin of the Arg/N-degron pathway, Analysis of higher plant N-degron pathway components and substrates via expression in S. cerevisiae, and so much more.

Cover image
Title page
Table of Contents
Series Page
Copyright
Contributors
Preface_ A nascent polypeptide emerges
Chapter One: Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells
Abstract
1: Introduction
2: Experimental design
3: General considerations
4: Cell growth, in vivo crosslinking, and cell lysis
5: Nuclease digestion and polysome profiling
6: Ribosome isolation for total translatome and selected translatome
7: Preparation of a ribosome footprint library
8: Data analysis
9: Prospects and conclusion
Acknowledgments
References
Chapter Two: Deformylation of nascent peptide chains on the ribosome
Abstract
1: Introduction
2: General methods
3: PDF purification
4: PDF substrates
5: RNC deformylation
6: PDF-ribosome interactions
7: Conclusion
Acknowledgments
References
Chapter Three: Optimizing purification and activity assays of N-terminal methyltransferase complexes
Abstract
1: Introduction
2: Design of single and dual expression constructs
3: Purification of recombinant protein
4: Western blot in vitro methyltransferase assays for full-length protein substrates
5: Luminescent in vitro methyltransferase assays for peptide substrates
6: Dual methyltransferase reactions for measuring inhibition/activation
7: Summary and conclusions
Acknowledgments
References
Chapter Four: Site-specific methylation on α-N-terminus of peptides through chemical and enzymatic methods
Abstract
1: Introduction
2: Preparation of recombinant NTMT1
3: Synthesis of Nα methylated peptides
4: Cleavage and purification of Nα methylated peptides
5: Summary and prospects
Acknowledgment
References
Chapter Five: Biochemical and structural analysis of N-myristoyltransferase mediated protein tagging
Abstract
1: Introduction
2: Materials, reagents, and buffers
3: Preparation of noncommercial N-myristoyl protein or CoA derivatives
4: Characterization of NMT-induced modifications on protein targets
5: Structural studies of NMT and its complexes with substrates and products
6: Conclusions and future prospects
Acknowledgments
Funding
References
Chapter Six: Kinetic and catalytic features of N-myristoyltransferases
Abstract
1: Introduction
2: Materials, reagents, and buffers
3: Kinetic analysis
4: Conclusions
Acknowledgments
Funding
References
Chapter Seven: Use of alkyne-tagged myristic acid to detect N-terminal myristoylation
Abstract
1: Introduction
2: Synthesis of Alk-12probes
3: Alk-12 and rhodamine labeling of targeted proteins in cells with click chemistry
4: Alk-12 and PEG-5000 labeling of targeted proteins
5: Global labeling of N-myristoylated proteins in whole cell lysate
6: Global profiling of N-myristoylated proteins using SILAC proteomics
References
Chapter Eight: Peptide CoA conjugates for in situ proteomics profiling of acetyltransferase activities
Abstract
1: Introduction
2: Peptide probe synthesis
3: Peptide immobilization and pulldowns
4: Filter-assisted sample preparation
5: LC-MS/MS and data analysis
6: Summary and outlook
Acknowledgments
References
Chapter Nine: A decoupled Virotrap approach to study the interactomes of N-terminal proteoforms
Abstract
1: Introduction
2: Design and creation of constructs for decoupled Virotrap
3: Western blot analysis as initial screen during decoupled Virotrap studies
4: An MS-readout for decoupled Virotrap samples
5: Data analysis
6: Results
7: Conclusions and discussion
Acknowledgments
Data availability
References
Chapter Ten: The amino-dipeptidyl peptidases DPP8 and DPP9: Purification and enzymatic assays
Abstract
1: Introduction
2: Purification of recombinant DPP8 and DPP9
3: Immunoprecipitation of FLAG tagged DPP8 or DPP9
4: DPP8 and DPP9 enzyme kinetics
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