In-Depth Characterization of Apoptosis N-terminome Reveals a Link Between Caspase-3 Cleavage and Post-Translational N-terminal Acetylation

Abstract

Proteins' N-termini contain information about their biochemical properties and functions, and they can undergo co- or post-translational modifications, as well as be processed by proteases. To expand the coverage of the N-terminome, we have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides. We applied LATE in combination with other N-terminomic method to study caspase-3 mediated proteolysis both in vitro and during apoptosis in cells. This enable us to identify many unreported caspase-3 cleavages, including some that cannot be identified by other methods. Furthermore, we find direct evidence that some of the caspase-3 generated neo-N-termini can be further modified by Nt-acetylation. This provides a global picture of the caspase-3 degradome and uncovers previously unrecognised functional crosstalk between post-translational Nt-acetylation and caspase proteolysis pathways.

Shifting the balance: soluble ADAM10 as a potential treatment for Alzheimer's disease

“…However, they do have some limitations that restrict the identification of potential of N-terminal peptides including the identification of neo-N-terminal peptides that were generated after proteolytic processing. To overcome this and expand the characterization of sADAM10 putative cleavage sites, we combined N-terminal enrichment (Chen et al, 2016 ; Weng et al, 2019 ) and C-terminal enrichment (Hanna et al, 2023 ). This combined approach provides a more comprehensive view of the putative sADAM10 substrates, as demonstrated for other proteases (van Damme et al, 2010 ).…”

“…The control samples were labeled with light formaldehyde (C 12 H 2 ), and the treated samples were labeled with heavy formaldehyde (C 13 D 2 ). Finally, the samples were divided, and half of each sample was taken to either N-terminal enrichment based on HYTANE (Chen et al, 2016 ; Weng et al, 2019 ) method using the protocol described in Hanna et al ( 2023 ) or C-terminal enrichment. Following enrichment and desalting, the samples were analyzed by tandem mass spectrometry using Thermo Q-Exactive Orbitrap HF coupled with Easy nano-LC 1000 capillary HPLC.…”

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

“…Many methods to isolate protein N- and C-terminal peptides have been developed. COFRADIC (combined fractional diagonal chromatography) (10) and TAILS (terminal amine isotopic labeling of substrates) (11) are widely known N-terminal peptide isolation methods, and other methods for N-terminal peptide isolation have also been reported (12). However, all of these methods require complex and time-consuming procedures involving chemical modifications to block amines, and the efficiency and selectivity of these chemical modifications are still problematic.…”