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DPP8/9 processing of human AK2 unmasks an IAP binding motif

ABSTRACT: AK2 is n-terminally processed by methionine aminopeptidases, N-terminal acetylation and DDP8/9 to become an active protein which is then sorted in the mitochondrial intermembrane space. We used peptide pull-downs of free and acetylated N-terminal peptides of AK2 from the different processing steps to identify binding partners by MS. We identified the inhibitor of apoptosis E3 ligase proteins (IAPs; BIRC2, 3, 6 and XIAP) as new binding partners for processed AK2 and show that they are responsible for AK2 degradation in the cytosol.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Tanja Bange

LAB HEAD: Tanja Bange

PROVIDER: PXD047178 | Pride | 2025-03-13

REPOSITORIES: Pride

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			Action	DRS
	20220317_Q2_U4_1-10_AB_SA_FM1052_10.raw	Raw
	20220317_Q2_U4_1-10_AB_SA_FM1052_12.raw	Raw
	20220317_Q2_U4_1-10_AB_SA_FM1052_14.raw	Raw
	20220317_Q2_U4_1-10_AB_SA_FM1052_16.raw	Raw
	20220317_Q2_U4_1-10_AB_SA_FM1052_18.raw	Raw

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Project description:Co-translational N-terminal (Nt-) acetylation of nascent polypeptides is catalyzed by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence is the major factor determining whether or not a given protein is Nt-acetylated. In humans, six different NATs, denoted NatA-NatF, are identified. In the current study we used N-terminal COFRADIC analysis to define the in vivo substrate specificity of Naa50 (Nat5)/NatE as this has long remained elusive. Three yeast strains were generated; a control strain endogenously expressing yNaa50, a deletion strain depleted of yNaa50 and a strain deleted of yNaa50 but ectopically expressing human Naa50. When comparing the Nt-acetylation status of different N-termini in the control strain with the deletion strain, a reduction in Nt-acetylation for several yeast proteins was observed. To our surprise, these substrates were not of the predicted NatE-type substrates, but rather canonical NatA substrates. Further, ectopic expression of hNaa50 mainly resulted in the Nt-acetylation of a selected class of otherwise Nt-free yeast N-termini besides increasing the degree of Nt-acetylation of several other yeast proteins, and as such (partially) complementing those N-termini displaying reduced Nt-acetylation upon yNAA50-deletion. The preferred substrates of hNaa50 were predominantly Met-starting N-termini including Met-Lys-, Met-Val-, Met-Ala-, Met-Tyr-, Met-Phe-, Met-Leu-, Met-Ser- and Met-Thr, and highly overlapped with the previously identified human Naa60/NatF substrate specificity profile. Identification of several hNaa50 substrates with a small amino acid in the second position also revealed a potential interplay between the NATs and methionine aminopeptidases (MetAPs). The initiator Methionine (iMet) is normally cleaved off by MetAPs when the second amino acid is small, but our in vitro data suggest that in contrast to a free iMet, an Nt-acetylated iMet is not hydrolyzed by MetAPs. Thus, Naa50-mediated Nt-acetylation may potentially act as a mechanism to retain the iMet of proteins with a small amino acid at the second position that normally would be hydrolyzed.

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DPP8/9 processing of human AK2 unmasks an IAP binding motif

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