Positional proteomics reveals differences in N‐terminal proteoform stability

Abstract

To understand the impact of alternative translation initiation on a proteome, we performed a proteome‐wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N‐terminal proteoforms of individual genes. By combining pulsed SILAC with N‐terminal COFRADIC, we monitored the stability of 1,941 human N‐terminal proteoforms, including 147 N‐terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. N‐terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N‐terminal amino acid identity or truncation length. We discovered that the removal of initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms, while susceptibility for N‐terminal acetylation did not seem to influence protein turnover rates. Taken together, our findings reveal differences in protein stability between N‐terminal proteoforms and point to a role for alternative translation initiation and co‐translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis.

Determinants and Regulation of Protein Turnover in Yeast

“…The median half-life was 2.18 h, similar to our previous study (Martin-Perez and Villen, 2015). This value also matches the cellular doubling-time (2.0 ± 0.1 h), in agreement with previous turnover studies in human cells (Boisvert et al, 2012; Gawron et al, 2016), suggesting that generally protein replacement is driven by dilution due to cell division.…”

“…The presence of certain amino acids in the N-terminus of a protein can be destabilizing and has been proposed as a predictor of the protein’s half-life (Bachmair et al, 1986; Gawron et al, 2016). Protein N-termini sequence analysis revealed a significant enrichment of alanine and serine residues in the position immediately adjacent to the initiator methionine on short-lived proteins (Figure S4A).…”

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes

“…In comparison, earlier pulse-chase SILAC studies in dividing cells reported less variable protein degradation. This may be because cell division is associated with activation of ubiquitin ligases (44) and higher rates of protein turnover (23)(24)(25)(26)(27)(28). In contrast to the effects of insulin on protein synthesis, insulin modulated the degradation of only 34 proteins using an adj.…”

“…Furthermore, the full complement of proteins that is synthesized and/or degraded in response to insulin has not been characterized. Here we investigated the global effects of hyperactive insulin signaling on protein synthesis and degradation using pulse-chase labeling with stable isotopes (stable isotope-labeled amino acids in cell culture (SILAC)) (23)(24)(25)(26)(27)(28) and high resolution mass spectrometry (MS)-based proteomics.…”

Simple, scalable, and ultrasensitive tip-based identification of protease substrates

“…Upon proteolytic cleavage as part of the common bottom-up proteomics strategy, this labelling allows quantifying N-terminal peptides but also distinguishing them from internal peptides with free Ntermini generated during in vitro digestion. Both methods have been used in numerous studies providing novel insights into proteases and their substrates (1,(8)(9)(10). Nevertheless, likely due to challenges in technical and (particularly in the past) data analysis aspects, the number of labs worldwide applying Nterminomics methods to screen for protease substrates is still limited.…”