Evaluating possible maternal effect lethality and genetic background effects in Naa10 knockout mice.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting approximately 80% of all human proteins. The human essential X-linked gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. There is extensive genetic variation in humans with missense, splice-site, and C-terminal frameshift variants in NAA10. In mice, Naa10 is not an essential gene, as there exists a paralogous gene, Naa12, that substantially rescues Naa10 knockout mice from embryonic lethality, whereas double knockouts (Naa10-/Y Naa12-/-) are embryonic lethal. However, the phenotypic variability in the mice is nonetheless quite extensive, including piebaldism, skeletal defects, small size, hydrocephaly, hydronephrosis, and neonatal lethality. Here we replicate these phenotypes with new genetic alleles in mice, but we demonstrate their modulation by genetic background and environmental effects. We cannot replicate a prior report of "maternal effect lethality" for heterozygous Naa10-/X female mice, but we do observe a small amount of embryonic lethality in the Naa10-/Y male mice on the inbred genetic background in this different animal facility.

Structural properties of the HNF-1A transactivation domain.

Hepatocyte nuclear factor 1α (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic ß-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic ß-cells.

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity.

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

Using cell lysates to assess N-terminal acetyltransferase activity and impairment.

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 14C-Ac-CoA and scintillation to detect the formation of Nt-acetylated peptide products.

NATs at a glance.

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.

Loss of N-terminal acetyltransferase A activity induces thermally unstable ribosomal proteins and increases their turnover in Saccharomyces cerevisiae.

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employ a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which causes global proteome remodeling. Pulsed-SILAC experiments reveals that NatA-deficient strains consistently increase degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovers decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.

Optimized bisubstrate inhibitors for the actin N-terminal acetyltransferase NAA80.

Acetylation of protein N-termini is one of the most common protein modifications in the eukaryotic cell and is catalyzed by the N-terminal acetyltransferase family of enzymes. The N-terminal acetyltransferase NAA80 is expressed in the animal kingdom and was recently found to specifically N-terminally acetylate actin, which is the main component of the microfilament system. This unique animal cell actin processing is essential for the maintenance of cell integrity and motility. Actin is the only known substrate of NAA80, thus potent inhibitors of NAA80 could prove as important tool compounds to study the crucial roles of actin and how NAA80 regulates this by N-terminal acetylation. Herein we describe a systematic study toward optimizing the peptide part of a bisubstrate-based NAA80 inhibitor comprising of coenzyme A conjugated onto the N-terminus of a tetrapeptide amide via an acetyl linker. By testing various combinations of Asp and Glu which are found at the N-termini of ß- and γ-actin, respectively, CoA-Ac-EDDI-NH2 was identified as the best inhibitor with an IC50 value of 120 nM.

A nonsense variant in the N-terminal acetyltransferase NAA30 may be associated with global developmental delay and tracheal cleft.

Most human proteins are N-terminally acetylated by N-terminal acetyltransferases (NATs), which play crucial roles in many cellular functions. The NatC complex, comprising the catalytic subunit NAA30 and the auxiliary subunits NAA35 and NAA38, is estimated to acetylate up to 20% of the human proteome in a co-translational manner. Several NAT enzymes have been linked to rare genetic diseases, causing developmental delay, intellectual disability, and heart disease. Here, we report a de novo heterozygous NAA30 nonsense variant c.244C>T (p.Q82*) (NM_001011713.2), which was identified by whole exome sequencing in a 5-year-old boy presenting with global development delay, autism spectrum disorder, hypotonia, tracheal cleft, and recurrent respiratory infections. Biochemical studies were performed to assess the functional impact of the premature stop codon on NAA30's catalytic activity. We find that NAA30-Q82* completely disrupts the N-terminal acetyltransferase activity toward a classical NatC substrate using an in vitro acetylation assay. This finding corresponds with structural modeling showing that the truncated NAA30 variant lacks the entire GNAT domain, which is required for catalytic activity. This study suggests that defective NatC-mediated N-terminal acetylation can cause disease, thus expanding the spectrum of NAT variants linked to genetic disease.

Novel biallelic variants expand the phenotype of NAA20-related syndrome.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

Protein Termini 2022: central roles of protein ends.

Although locating at the protein ends, N- and C-termini are at the center of numerous cellular functions. This topic engages an increasing number of scientists, recently forming the International Society of Protein Termini (ISPT). Protein Termini 2022 gathered this interdisciplinary community to discuss how protein ends may steer protein functionality.

Actin finally matures: uncovering machinery and impact.

Actin, one of the most abundant proteins in nature and a key component of the cytoskeleton, undergoes a unique multistep N-terminal (Nt) maturation. In a recent report, Haahr et al. identified actin maturation protease (ACTMAP) as the dedicated actin aminopeptidase and showed that its absence is associated with abnormal muscle physiology.

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC.

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

Structural and biophysical characterization of transcription factor HNF-1A as a tool to study MODY3 diabetes variants.

Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating the expression of numerous target genes. Pathogenic variants in the HNF1A gene are known to cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), a disease characterized by dominant inheritance, age of onset before 25 to 35 years of age, and pancreatic ß-cell dysfunction. A precise diagnosis can alter management of this disease, as insulin can be exchanged with sulfonylurea tablets and genetic counseling differs from polygenic forms of diabetes. Therefore, more knowledge on the mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants is required for precise diagnostics. Here, we structurally and biophysically characterized an HNF-1A protein containing both the DNA-binding domain and the dimerization domain, and determined the folding and DNA-binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S). All three variants showed reduced functionality compared to the WT protein. Furthermore, while the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, we found the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for these investigated variants and present a novel approach for the dissection of structurally unstable and DNA-binding defective variants. This study indicates that structural and biochemical investigation of HNF-1A is a valuable tool in reliable variant classification needed for precision diabetes diagnostics and management.

Biochemical analysis of novel NAA10 variants suggests distinct pathogenic mechanisms involving impaired protein N-terminal acetylation.

NAA10 is the catalytic subunit of the N-terminal acetyltransferase complex, NatA, which is responsible for N-terminal acetylation of nearly half the human proteome. Since 2011, at least 21 different NAA10 missense variants have been reported as pathogenic in humans. The clinical features associated with this X-linked condition vary, but commonly described features include developmental delay, intellectual disability, cardiac anomalies, brain abnormalities, facial dysmorphism and/or visual impairment. Here, we present eight individuals from five families with five different de novo or inherited NAA10 variants. In order to determine their pathogenicity, we have performed biochemical characterisation of the four novel variants c.16G>C p.(A6P), c.235C>T p.(R79C), c.386A>C p.(Q129P) and c.469G>A p.(E157K). Additionally, we clinically describe one new case with a previously identified pathogenic variant, c.384T>G p.(F128L). Our study provides important insight into how different NAA10 missense variants impact distinct biochemical functions of NAA10 involving the ability of NAA10 to perform N-terminal acetylation. These investigations may partially explain the phenotypic variability in affected individuals and emphasise the complexity of the cellular pathways downstream of NAA10.

The Final Maturation State of ß-actin Involves N-terminal Acetylation by NAA80, not N-terminal Arginylation by ATE1.

Actin is a hallmark protein of the cytoskeleton in eukaryotic cells, affecting a range of cellular functions. Actin dynamics is regulated through a myriad of actin-binding proteins and post-translational modifications. The mammalian actin family consists of six different isoforms, which vary slightly in their N-terminal (Nt) sequences. During and after synthesis, actins undergo an intricate Nt-processing that yields mature actin isoforms. The ubiquitously expressed cytoplasmic ß-actin is Nt-acetylated by N-alpha acetyltransferase 80 (NAA80) yielding the Nt-sequence Ac-DDDI-. In addition, ß-actin was also reported to be Nt-arginylated by arginyltransferase 1 (ATE1) after further peptidase-mediated processing, yielding RDDI-. To characterize in detail the Nt-processing of actin, we used state-of-the-art proteomics. To estimate the relative cellular levels of Nt-modified proteoforms of actin, we employed NAA80-lacking cells, in which actin was not Nt-acetylated. We found that targeted proteomics is superior to a commercially available antibody previously used to analyze Nt-arginylation of ß-actin. Significantly, despite the use of sensitive mass spectrometry-based techniques, we could not confirm the existence of the previously claimed Nt-arginylated ß-actin (RDDI-) in either wildtype or NAA80-lacking cells. A very minor level of Nt-arginylation of the initially cleaved ß-actin (DDDI-) could be identified, but only in NAA80-lacking cells, not in wildtype cells. We also identified small fractions of cleaved and unmodified ß-actin (DDI-) as well as cleaved and Nt-acetylated ß-actin (Ac-DDI-). In sum, we show that the multi-step Nt-maturation of ß-actin is terminated by NAA80, which Nt-acetylates the exposed Nt-Asp residues, in the virtual absence of previously claimed Nt-arginylation.

Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells.

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-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.

Naa12 compensates for Naa10 in mice in the amino-terminal acetylation pathway.

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.