Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and Human Methionine Aminopeptidases

Abstract

Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of methionyl removal from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1' residue, but their specificity at positions P2' and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1' position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently two orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2' to P5', all three MetAPs have broad specificity, but are poorly active toward peptides containing a proline at the P2' position. In addition, the human MetAPs disfavor acidic residues at the P2' to P5' positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins. KeywordsMethionine aminopeptidase; N-terminal processing; substrate specificity; kinetics; peptide library Ribosomal protein synthesis is universally initiated with methionine (in eukaryotic cytoplasm) or N-formylmethionine (in prokaryotes, mitochondria, and chloroplasts). During protein maturation, the N-formyl group is removed by peptide deformylase, leaving methionine with a free NH 2 group (1). Subsequently, the initiator methionine is removed from many but not all of the proteins by methionine aminopeptidase(s) (MetAPs). For example, in a cytosolic extract of Escherichia coli, only 40% of the polypeptides retain the initiator methionine, whereas the majority of the polypeptides display alanine, serine, or threonine at their N-termini (2). There are two types of MetAPs, MetAP1 and MetAP2.

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…Then the observed partial NME could be linked to the type of human cell lines that were used in this study. If it is not the case, this further supports the fact that MetAP2, the enzyme in charge of this cleavage, is more limiting in humans than in plants (71). This could explain why it is possible to inactivate this enzyme in plants and not in animals (72,73).…”

Group VII Ethylene Response Factors in Arabidopsis: Regulation and Physiological Roles

“…Initial recognition of plant ERF-VII proteins as potential oxygen-sensitive substrates entailed the finding that this consensus contains a redox-sensitive Cys, in such a position as to be amenable to recognition by a specialized proteolytic pathway, known as the Arg-Cys/N-end rule pathway (NERP) for proteasomal degradation (Gibbs et al, 2011;Licausi et al, 2011a). A sequence of orderly reactions involves ERF-VII proteins (Box 1; Graciet et al, 2009;Tasaki et al, 2012;Xiao et al, 2010). Mature ERF-VII proteins expose an N-terminal Cys (Cys2), which functions as a degradation signature ("N-degron") targeting these proteins to the 26S proteasome, upon sequential recruitment of arginyl transferase and E3 ubiquitin ligase enzymes (Bachmair et al, 1986;Varshavsky 2011).…”

Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway

“…The start (AUG) codon-encoded N-terminal Met residue of nascent proteins is cotranslationally cleaved off by ribosomeassociated Met-aminopeptidases (MetAPs) if a residue at position 2, to be made N-terminal by the cleavage, is not larger than Val (41,74). Thus, for example, the Met residue of the Nterminal Met-Leu-Ser sequence of wild-type rat AANAT (denoted as MLS rAANAT) (Fig.…”

“…In contrast, the N-terminal Met residue of the mutant N-terminal sequence (Met)-Pro-Leu-Ser of (M)PLS rAANAT 3f would be cotranslationally cleaved off by MetAPs. The resulting N-terminal Pro is not Nt-acetylated, at least in S. cerevisiae, and is usually not Nt-acetylated in mammalian cells as well (59,63 (41,74). However, in contrast to the resulting N-terminal Ser of wild-type ST hAANAT 3f , which would be expected to be cotranslationally Nt-acetylated, the N-terminal Pro of the mutant PT hAANAT 3f is not Nt-acetylated, at least in S. cerevisiae, and is usually not Nt-acetylated in mammalian cells as well (59,63).…”