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NAA15 haploinsufficiency can cause congenital heart disease and perturbs protein levels in induced pluripotent stem cells

ABSTRACT: Rationale- NAA15 is a component of the N-terminal (Nt) acetyltransferase complex, NatA. The mechanism by which NAA15 haploinsufficiency causes congenital heart disease (CHD) remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective- We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity, and identifying proteins dependent upon a full amount of NAA15.Methods and Results - We introduced heterozygous LoF, compound heterozygous and missense residues (R276W) in iPS cells using CRISPR/Cas9. Haploinsufficient NAA15 iPS cells differentiate into cardiomyocytes, unlike NAA15-null iPS cells, presumably due to altered composition of NatA. Mass spectrometry (MS) analyses reveal ~80% of identified iPS cell NatA targeted proteins displayed partial or complete Nt-acetylation. Between null and haploinsufficient NAA15 cells Nt-acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W iPSCs. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; eighteen were ribosomal-associated proteins. At least four proteins were encoded by genes known to cause autosomal dominant CHD. Conclusions - These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and Nt-acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated CHD. Additionally, genetically engineered iPS cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE160809 | GEO | 2020/11/05

REPOSITORIES: GEO

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Project description:N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.

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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NAA15 haploinsufficiency can cause congenital heart disease and perturbs protein levels in induced pluripotent stem cells

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