Protein termini represent a major route to protein regulation. From the moment the very first amino acid of a polypeptide chain exits the ribosome there is potential for steering from the cellular environment. This volume of Methods in Enzymology Modifications and Targeting of Protein Termini focuses on Protein N-termini and C-termini and their modifications which include acetylation, arginylation, myristoylation and oxidation. Also, the impact of terminal modifications is covered, in particular the impact on protein turnover and the ubiquitin E3 ligases which specifically recognize protein N-termini (N-degrons) and C-termini (C-degrons). In addition to the detailed methods and laboratory protocols, the chapters include informative overviews and reviews of the different subfields.
1. In vitro acetyltransferase assays for NAT enzymes
Ronen Marmorstein
2. A fluorescent CPM-based in vitro acetylation assay: a tool for assessing
N-Terminal acetyltransferase activity and profiling compound activity
Tanja Bange
3. Methods for Purification and Activity Analysis of N-Terminal
Acetyltransferases
Rong Huang
4. N-terminal acetylation of plastid precursor proteins
Sacha Baginsky
5. Utilizing N-terminal acetylation specific antibodies
Henriette Aksnes
6. Functional assessment of N-terminal acetyltransferase variants
Nina McTiernan
7. Affinity purification-mass spectrometry to identify nuclear protein
interactions of N-terminal acetyltransferase NAA40
Antonis Kirmizis
8. A pan-N-formylmethionine-specific antibody as a tool to for analyzing
N-terminal formylation
Cheol-Sang Hwang
9. Chemical proteomic approaches to investigate N-myristoylation
Edward W. Tate
10. Analysis of the oxidation state of N-terminal methionines
Sina Ghaemmaghami
11. A biophysical approach to studying N-terminal cysteine oxidase substrate
preferences
Mark White
12. Recombinant expression, purification, and characterization of human ATE1
arginyltransferase
Yi Zhang
13. In-bacteria arginylation assay
Yi Zhang
14. Identification of arginylated N-termini with specific antibodies
Jasper Eising
15. Characterization of the autophagic N-degron pathway and monitoring its
chemical modulation for therapeutic development
Yong Tae Kwon
16. Developing a flow cytometric method to evaluate the stability of protein
N-termini
Aditya M. Kunjapur
17. Degronopedia: A practical guide to identifying and targeting protein
degrons
Wojciech Pokrzywa
18. Generation and usage of ubiquitin-reference technique (URT) plasmids to
validate N-degrons
Chang Hoon Ji
19. Quantitative Insights into Protein Turnover and Ubiquitination with HiBiT
and NanoBRET
Wojciech Pokrzywa
20. Characterization of the autophagy E3 ligase/N-recognin KCMF1
Chang Hoon Ji
21. Identification of Ac/N-degron-recognition domain within the MARCHF6 E3
ubiquitin ligase
Cheol-Sang Hwang
22. TurboID technique for proximity labelling of interacting proteins
Greta Jarck
23. Proteome analysis of nascent polypeptide chains using puromycin
Koshi Imami
24. HUNTER-DIA: An updated protocol for enrichment and mass
spectrometry-based identification of protein N-termini
Pitter F. Huesgen
25. TERMINER - Bioinformatic detection and annotation of proteolytic protein
termini in shotgun proteomics data
Oliver Schilling
26. Chemical proteomic approaches to investigate S-prenylation
Edward W. Tate
27. Quantitative analysis of C-terminal prenylated protein levels using
tandem mass tagging
Mark Distefano
28. Optimizing purification and FP-based binding assays for the E3 ligase
FEM1C
Rong Huang
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.
