The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE ...co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.
The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits-the catalytic ...subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While
c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity,
c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.
Abstract Oxygen homeostasis is maintained in plants and animals by O 2 -sensing enzymes initiating adaptive responses to low O 2 (hypoxia). Recently, the O 2 -sensitive enzyme ADO was shown to ...initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O 2 -dependent protein stability and the hypoxic response.
NAA10 is a major
-terminal acetyltransferase (NAT) that catalyzes the cotranslational
-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) ...activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.
NAA10 is the catalytic subunit of the major N-terminal acetyltransferase complex NatA which acetylates almost half the human proteome. Over the past decade, many NAA10 missense variants have been ...reported as causative of genetic disease in humans. Individuals harboring NAA10 variants often display variable degrees of intellectual disability (ID), developmental delay, and cardiac anomalies. Initially, carrier females appeared to be oligo- or asymptomatic with X-inactivation pattern skewed towards the wild type allele. However, recently it has been shown that NAA10 variants can cause syndromic or non-syndromic intellectual disability in females as well. The impact of specific NAA10 variants and the X-inactivation pattern on the individual phenotype in females remains to be elucidated.
Here we present a novel de novo NAA10 (NM_003491.3) c.47A > C;= (p.His16Pro;=) variant identified in a young female. The 10-year-old girl has severely delayed motor and language development, disturbed behavior with hyperactivity and restlessness, moderate dilatation of the ventricular system and extracerebral CSF spaces. Her blood leukocyte X-inactivation pattern was skewed (95/5) towards the maternally inherited X-chromosome. Our functional study indicates that NAA10 p.(H16P) impairs NatA complex formation and NatA catalytic activity, while monomeric NAA10 catalytic activity appears to be intact. Furthermore, cycloheximide experiments show that the NAA10 H16P variant does not affect the cellular stability of NAA10.
We demonstrate that NAA10 p.(His16Pro) causes a severe form of syndromic ID in a girl most likely through impaired NatA-mediated Nt-acetylation of cellular proteins. X-inactivation analyses showed a skewed X-inactivation pattern in DNA from blood of the patient with the maternally inherited allele being preferentially methylated/inactivated.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The NAA10-NAA15 (NatA) protein complex is an N-terminal acetyltransferase responsible for acetylating ~ 40% of eukaryotic proteins. In recent years, NAA10 variants have been found in patients with an ...X-linked developmental disorder called Ogden syndrome in its most severe form and, in other familial or de novo cases, with variable degrees of syndromic intellectual disability (ID) affecting both sexes.
Here we report and functionally characterize a novel and de novo NAA10 (NM_003491.3) c.332 T > G p.(V111G) missense variant, that was detected by trio-based whole exome sequencing in an 11 year old girl with mild/moderate non-syndromic intellectual disability. She had delayed motor and language development, but normal behavior without autistic traits. Her blood leukocyte X-inactivation pattern was within normal range (80/20). Functional characterization of NAA10-V111G by cycloheximide chase experiments suggests that NAA10-V111G has a reduced stability compared to NAA10-WT, and in vitro acetylation assays revealed a reduced enzymatic activity of monomeric NAA10-V111G but not for NAA10-V111G in complex with NAA15 (NatA enzymatic activity).
We show that NAA10-V111G has a reduced stability and monomeric catalytic activity, while NatA function remains unaltered. This is the first example of isolated NAA10 dysfunction in a case of ID, suggesting that the syndromic cases may also require a degree of compromised NatA function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40–50% of all ...mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking
Naa10
show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather
Naa10
nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism, and urogenital anomalies.
Naa12
is a previously unannotated
Naa10
-like paralog with NAT activity that genetically compensates for
Naa10
. Mice deficient for
Naa12
have no apparent phenotype, whereas mice deficient for
Naa10
and
Naa12
display embryonic lethality. The discovery of
Naa12
adds to the currently known machinery involved in amino-terminal acetylation in mice.
NATs at a glance Aksnes, Henriette; McTiernan, Nina; Arnesen, Thomas
Journal of cell science,
2023-Jul-15, Letnik:
136, Številka:
14
Journal Article
Recenzirano
Odprti dostop
Most proteins receive an acetyl group at the N terminus while in their nascency as the result of modification by co-translationally acting N-terminal acetyltransferases (NATs). The N-terminal acetyl ...group can influence several aspects of protein functionality. From studies of NAT-lacking cells, it is evident that several cellular processes are affected by this modification. More recently, an increasing number of genetic cases have demonstrated that N-terminal acetylation has crucial roles in human physiology and pathology. In this Cell Science at a Glance and the accompanying poster, we provide an overview of the human NAT enzymes and their properties, substrate coverage, cellular roles and connections to human disease.
The vast majority of eukaryotic proteins are subjected to N-terminal (Nt) acetylation. This reaction is catalyzed by a group of N-terminal acetyltransferases (NATs), which co- or post-translationally ...transfer an acetyl group from Acetyl coenzyme A to the protein N-terminus. Nt-acetylation plays an important role in many cellular processes, but the functional consequences of this widespread protein modification are still undefined for most proteins. Several in vitro acetylation assays have been developed to study the catalytic activity and substrate specificity of NATs or other acetyltransferases. These assays are valuable tools that can be used to define substrate specificities of yet uncharacterized NAT candidates, assess catalytic impairment of pathogenic NAT variants, and determine the potency of chemical inhibitors. The enzyme input in acetylation assays is typically acetyltransferases that have been recombinantly expressed and purified or immunoprecipitated proteins. In this chapter, we highlight how cell lysates can also be used to assess NAT catalytic activity and impairment when used as input in a previously described isotope-based in vitro Nt-acetylation assay. This is a fast and highly sensitive method that utilizes isotope labeled
C-Ac-CoA and scintillation to detect the formation of Nt-acetylated peptide products.
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