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.

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.

Forward genetic screening identifies novel roles for N-terminal acetyltransferase C and histone deacetylase in C. elegans development.

Coordinating the balance between development and stress responses is critical for organismal survival. However, the cellular signaling controlling this mechanism is not well understood. In Caenorhabditis elegans, it has been hypothesized that a genetic network regulated by NIPI-3/Tibbles may control the balance between animal development and immune response. Using a nipi-3(0) lethality suppressor screen in C. elegans, we reveal a novel role for N-terminal acetyltransferase C complex natc-1/2/3 and histone deacetylase hda-4, in the control of animal development. These signaling proteins act, at least in part, through a PMK-1 p38 MAP kinase pathway (TIR-1-NSY-1-SEK-1-PMK-1), which plays a critical role in the innate immunity against infection. Additionally, using a transcriptional reporter of SEK-1, a signaling molecule within this p38 MAP kinase system that acts directly downstream of C/EBP bZip transcription factor CEBP-1, we find unexpected positive control of sek-1 transcription by SEK-1 along with several other p38 MAP kinase pathway components. Together, these data demonstrate a role for NIPI-3 regulators in animal development, operating, at least in part through a PMK-1 p38 MAPK pathway. Because the C. elegans p38 MAP kinase pathway is well known for its role in cellular stress responses, the novel biological components and mechanisms pertaining to development identified here may also contribute to the balance between stress response and development.

Human NAA30 can rescue yeast mak3∆ mutant growth phenotypes.

N-terminal acetylation is an irreversible protein modification that primarily occurs co-translationally, and is catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). The NatC complex (NAA30-NAA35-NAA38) is a major NAT enzyme, which was first described in yeast and estimated to N-terminally acetylate ∼20% of the proteome. The activity of NatC is crucial for the correct functioning of its substrates, which include translocation to the Golgi apparatus, the inner nuclear membrane as well as proper mitochondrial function. We show in comparative viability and growth assays that yeast cells lacking MAK3/NAA30 grow poorly in non-fermentable carbon sources and other stress conditions. By using two different experimental approaches and two yeast strains, we show that liquid growth assays are the method of choice when analyzing subtle growth defects, keeping loss of information to a minimum. We further demonstrate that human NAA30 can functionally replace yeast MAK3/NAA30. However, this depends on the genetic background of the yeast strain. These findings indicate that the function of MAK3/NAA30 is evolutionarily conserved from yeast to human. Our yeast system provides a powerful approach to study potential human NAA30 variants using a high-throughput liquid growth assay with various stress conditions.

Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates.

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30-Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure-function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins.

A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity.

The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.

Evaluation of Salivary Exosomal Chimeric GOLM1-NAA35 RNA as a Potential Biomarker in Esophageal Carcinoma.

PURPOSE: Transcriptionally induced chimeric RNAs are an important emerging area of research into molecular signatures for biomarker and therapeutic target development. Salivary exosomes represent a relatively unexplored, but convenient, and noninvasive area of cancer biomarker discovery. However, the potential of cancer-derived exosomal chimeric RNAs in saliva as biomarkers is unknown. Here, we explore the potential clinical utility of salivary exosomal GOLM1-NAA35 chimeric RNA (seG-NchiRNA) in esophageal squamous cell carcinoma (ESCC). EXPERIMENTAL DESIGN: In a retrospective study, the prognostic significance of G-NchiRNA was determined in ESCC tissues. The correlation between seG-NchiRNA and circulating exosomal or tumoral G-NchiRNA was ascertained in cultured cells and mice. In multiple prospective cohorts of patients with ESCC, seG-NchiRNA was measured by qRT-PCR and analyzed for diagnostic accuracy, longitudinal monitoring of treatment response, and prediction of progression-free survival (PFS). RESULTS: Exosomal G-NchiRNA was readily detectable in ESCC cells and nude mouse ESCC xenografts. SeG-NchiRNA levels reflected tumor burden in vivo and correlated with tumor G-NchiRNA levels. In prospective studies of a training cohort (n = 220) and a validation cohort (n = 102), seG-NchiRNA levels were substantially reduced after ESCC resection. Moreover, seG-NchiRNA was successfully used to evaluate chemoradiation responsiveness, as well as to detect disease progression earlier than imaging studies. Changes in seG-NchiRNA levels also predicted PFS of patients after chemoradiation. CONCLUSIONS: SeG-NchiRNA constitutes an effective candidate noninvasive biomarker for the convenient, reliable assessment of therapeutic response, recurrence, and early detection.

Identification of an alternatively spliced nuclear isoform of human N-terminal acetyltransferase Naa30.

N-terminal acetylation is a highly abundant and important protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs). In humans, six different NATs have been identified (NatA-NatF), each composed of individual subunits and acetylating a distinct set of substrates. Along with most NATs, NatC acts co-translationally at the ribosome. The NatC complex consists of the catalytic subunit Naa30 and the auxiliary subunits Naa35 and Naa38, and can potentially Nt-acetylate cytoplasmic proteins when the initiator methionine is followed by a bulky hydrophobic/amphipathic residue at position 2. Here, we have identified a splice variant of human NAA30, which encodes a truncated protein named Naa30288. The splice variant was abundantly present in thyroid cancer tissues and in several different human cancer cell lines. Surprisingly, Naa30288 localized predominantly to the nucleus, as opposed to annotated Naa30 which has a cytoplasmic localization. Full-length Naa30 acetylated a classical NatC substrate peptide in vitro, whereas no significant NAT activity was detected for Naa30288. Due to the nuclear localization, we also examined acetyltransferase activity towards lysine residues. Neither full-length Naa30 nor Naa30288 displayed any lysine acetyltransferase activity. Overexpression of full-length Naa30 increased cell viability via inhibition of apoptosis. In contrast, Naa30288 did not exert an anti-apoptotic effect. In sum, we identified a novel and widely expressed Naa30 isoform with a potential non-catalytic role in the nucleus.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes.

Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization.

The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to-trans-Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis-Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization.

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity.

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.

Inter-individual variability and genetic influences on cytokine responses to bacteria and fungi.

Little is known about the inter-individual variation of cytokine responses to different pathogens in healthy individuals. To systematically describe cytokine responses elicited by distinct pathogens and to determine the effect of genetic variation on cytokine production, we profiled cytokines produced by peripheral blood mononuclear cells from 197 individuals of European origin from the 200 Functional Genomics (200FG) cohort in the Human Functional Genomics Project (http://www.humanfunctionalgenomics.org), obtained over three different years. We compared bacteria- and fungi-induced cytokine profiles and found that most cytokine responses were organized around a physiological response to specific pathogens, rather than around a particular immune pathway or cytokine. We then correlated genome-wide single-nucleotide polymorphism (SNP) genotypes with cytokine abundance and identified six cytokine quantitative trait loci (QTLs). Among them, a cytokine QTL at the NAA35-GOLM1 locus markedly modulated interleukin (IL)-6 production in response to multiple pathogens and was associated with susceptibility to candidemia. Furthermore, the cytokine QTLs that we identified were enriched among SNPs previously associated with infectious diseases and heart diseases. These data reveal and begin to explain the variability in cytokine production by human immune cells in response to pathogens.

A de novo interstitial deletion of 7q31.2q31.31 identified in a girl with developmental delay and hearing loss.

We report on a 4-year-old female who presented with unilateral sensorineural hearing loss and a concern for developmental delay. A genome-wide SNP array analysis was performed and revealed a de novo 3.2 Mb interstitial deletion of chromosome 7q31.2q31.31. This region contains thirteen protein-encoding genes. It is unknown whether haploinsufficiency of any of these genes is responsible for the clinical features of our patient. We reviewed, the clinical phenotype of a previously published 7q31.3 deletion patient and 18 additional patients with overlapping 7q31 deletions listed in the DECIPHER database. The most consistent feature in these patients and our proband is delayed speech and language development. Hearing loss is presented both in our proband and the published 7q31.3 patient. Our study suggests that a small region on chromosome 7q31.3 encompassing four genes, CFTR, CTTNBP2, NAA38, and ANKRD7, may represent a new locus for congenital hearing loss and/or speech development. © 2016 Wiley Periodicals, Inc.

Knockdown of NAT12/NAA30 reduces tumorigenic features of glioblastoma-initiating cells.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets. METHODS: Microarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation. RESULTS: While decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1α, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures. CONCLUSION: NAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1α), levels of p-MTOR (Ser2448) and the p53 pathway.

The DAF-16 FOXO transcription factor regulates natc-1 to modulate stress resistance in Caenorhabditis elegans, linking insulin/IGF-1 signaling to protein N-terminal acetylation.

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.

Aberrant chimeric RNA GOLM1-MAK10 encoding a secreted fusion protein as a molecular signature for human esophageal squamous cell carcinoma.

It is increasingly recognized that chimeric RNAs may exert a novel layer of cellular complexity that contributes to oncogenesis and cancer progression, and could be utilized as molecular biomarkers and therapeutic targets. To date yet no fusion chimeric RNAs have been identified in esophageal cancer, the 6th most frequent cause of cancer death in the world. While analyzing the expression of 32 recurrent cancer chimeric RNAs in esophageal squamous cell carcinoma (ESCC) from patients and cancer cell lines, we identified GOLM1-MAK10, as a highly cancer-enriched chimeric RNA in ESCC. In situ hybridization revealed that the expression of the chimera is largely restricted to cancer cells in patient tumors, and nearly undetectable in non-neoplastic esophageal tissue from normal subjects. The aberrant chimera closely correlated with histologic differentiation and lymph node metastasis. Furthermore, we demonstrate that chimera GOLM1-MAK10 encodes a secreted fusion protein. Mechanistic studies reveal that GOLM1-MAK10 is likely derived from transcription read-through/splicing rather than being generated from a fusion gene. Collectively, these findings provide novel insights into the molecular mechanism involved in ESCC and provide a novel potential target for future therapies. The secreted fusion protein translated from GOLM1-MAK10 could also serve as a unique protein signature detectable by standard non-invasive assays. These observations are critical as there is no clinically useful molecular signature available for detecting this deadly disease or monitoring the treatment response.