RESUMEN
During recovery from heat stress, plants clear away the heat-stress-induced misfolded proteins through the ubiquitin-proteasome system (UPS). In the UPS, the recognition of substrate proteins by E3 ligase can be regulated by the N-terminal acetyltransferase A (NatA) complex. Here, we determined that Arabidopsis STRESS-RELATED UBIQUITIN-ASSOCIATED-DOMAIN PROTEIN FACTOR 1 (SUF1) interacts with the NatA complex core subunit NAA15 and positively regulates NAA15. The suf1 and naa15 mutants are sensitive to heat stress; the NatA substrate N SNC1 is stabilized in suf1 mutant plants during heat stress recovery. Therefore, SUF1 and its interactor NAA15 play important roles in basal thermotolerance in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Termotolerancia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Termotolerancia/genética , Ubiquitinas/metabolismoRESUMEN
N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.
Asunto(s)
Acetiltransferasa A N-Terminal/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Acetilación , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/genética , Regiones Promotoras Genéticas , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Ribonucleoproteínas/metabolismo , Transducción de Señal , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.
Asunto(s)
Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetilación , Catálisis , Quinasas Ciclina-Dependientes/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Metabolismo de los Lípidos , Acetiltransferasa A N-Terminal/genética , Proteoma , Proteómica/métodos , Especificidad por SustratoRESUMEN
Nearly half of all human proteins are acetylated at their N-termini by the NatA N-terminal acetyltransferase complex. NAA10 is evolutionarily conserved as the catalytic subunit of NatA in complex with NAA15, but may also have NatA-independent functions. Several NAA10 variants are associated with genetic disorders. The phenotypic spectrum includes developmental delay, intellectual disability, and cardiac abnormalities. Here, we have identified the previously undescribed NAA10 c.303C>A and c.303C>G p.(N101K) variants in two unrelated girls. These girls have developmental delay, but they both also display hemihypertrophy a feature normally not observed or registered among these cases. Functional studies revealed that NAA10 p.(N101K) is completely impaired in its ability to bind NAA15 and to form an enzymatically active NatA complex. In contrast, the integrity of NAA10 p.(N101K) as a monomeric acetyltransferase is intact. Thus, this NAA10 variant may represent the best example of the impact of NatA mediated N-terminal acetylation, isolated from other potential NAA10-mediated cellular functions and may provide important insights into the phenotypes observed in individuals expressing pathogenic NAA10 variants.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Síndrome de Proteo/genética , Acetilación , Secuencia de Aminoácidos , Animales , Preescolar , Femenino , Células HeLa , Humanos , Discapacidad Intelectual/genética , Ratones , Modelos Moleculares , Mutación , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal/química , Fenotipo , Conformación Proteica , Síndrome de Proteo/diagnóstico por imagen , Ratas , Alineación de Secuencia , Levaduras , Pez CebraRESUMEN
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 NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 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.
Asunto(s)
Mutación/genética , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/genética , Acetilación , Secuencia de Aminoácidos , Biocatálisis , Células HeLa , Humanos , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/químicaRESUMEN
We present a male patient born at 38-wk gestation with rhizomelic shortening of extremities, hepatomegaly, ventriculomegaly, heart failure, severely depressed left ventricular function, biventricular hypertrophy, and biatrial enlargement. Additional physical findings included anteriorly displaced anus, vertebral anomalies, and brachydactyly. The patient's cardiac malformations led to persistent hypotension, sinus tachycardia, and multiorgan failure in the absence of arrhythmias. Rapid whole-exome sequencing was ordered on day of life (DOL) 8. The patient's family elected to withdraw supportive care, and he passed away that evening. Whole-exome sequencing returned posthumously and identified a variant in NAA10, E100K. The genotype-phenotype was closest to Ogden syndrome or amino-terminal acetyltransferase deficiency. Typical features of this rare X-linked syndrome include progeroid appearance, failure to thrive, developmental delays, hypotonia, and cardiac arrhythmias. Other family members were tested and the patient's mother, who has a history of mild intellectual disability, as well as a daughter born later, were identified as carriers. All carriers showed no cardiac findings. The carrier sister has manifested developmental delay and cortical atrophy. Protein modeling, evolution, dynamics, population variant assessments, and immunoprecipitation depict the deleterious nature of the variant on the interactions of NAA10 with NAA15 These findings had subsequent implications for posthumous diagnosis of the index patient, for female carriers, and regarding family planning. We highlight how these rapid genetic tests and variant characterization can potentially lead to informed decision-making between health-care providers and family members of patients with critical or lethal conditions when treatment options are limited.
Asunto(s)
Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Femenino , Genes Ligados a X , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Hepatomegalia/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Modelos Moleculares , Mutación , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal/química , Linaje , Taquicardia Sinusal , Secuenciación del ExomaRESUMEN
BACKGROUND: 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. CASE PRESENTATION: 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. DISCUSSION AND CONCLUSIONS: 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.
Asunto(s)
Discapacidad Intelectual/genética , Mutación/genética , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Inactivación del Cromosoma X/genética , Secuencia de Aminoácidos , Biocatálisis , Niño , Cicloheximida/metabolismo , Femenino , Células HeLa , Heterocigoto , Humanos , Masculino , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal/química , Linaje , SíndromeRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/metabolismo , Acetilación , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Acetiltransferasa A N-Terminal/antagonistas & inhibidores , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/antagonistas & inhibidores , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Relación Estructura-ActividadRESUMEN
BACKGROUND: N-terminal acetylation is a common protein modification in human cells and is catalysed by N-terminal acetyltransferases (NATs), mostly cotranslationally. The NAA10-NAA15 (NatA) protein complex is the major NAT, responsible for acetylating ~ 40% of human proteins. Recently, NAA10 germline variants were found in patients with the X-linked lethal Ogden syndrome, and in other familial or de novo cases with variable degrees of developmental delay, intellectual disability (ID) and cardiac anomalies. METHODS: Here we report a novel NAA10 (NM_003491.3) c.248G > A, p.(R83H) missense variant in NAA10 which was detected by whole exome sequencing in two unrelated boys with intellectual disability, developmental delay, ADHD like behaviour, very limited speech and cardiac abnormalities. We employ in vitro acetylation assays to functionally test the impact of this variant on NAA10 enzyme activity. RESULTS: Functional characterization of NAA10-R83H by in vitro acetylation assays revealed a reduced enzymatic activity of monomeric NAA10-R83H. This variant is modelled to have an altered charge density in the acetyl-coenzyme A (Ac-CoA) binding region of NAA10. CONCLUSIONS: We show that NAA10-R83H has a reduced monomeric catalytic activity, likely due to impaired enzyme-Ac-CoA binding. Our data support a model where reduced NAA10 and/or NatA activity cause the phenotypes observed in the two patients.
Asunto(s)
Acetiltransferasas/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Mutación Missense , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Acetilación , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Preescolar , Humanos , Lactante , Masculino , Modelos Moleculares , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/metabolismo , Fenotipo , Dominios Proteicos , Homología de Secuencia de Aminoácido , Secuenciación del ExomaRESUMEN
NatA co-translationally acetylates the N termini of over 40% of eukaryotic proteins and can associate with another catalytic subunit, Naa50, to form a ternary NatA/Naa50 dual enzyme complex (also called NatE). The molecular basis of association between Naa50 and NatA and the mechanism for how their association affects their catalytic activities in yeast and human are poorly understood. Here, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We find that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. These studies have implications for understanding the role of the NatA/Naa50 complex in modulating the majority of the N-terminal acetylome in diverse species.
Asunto(s)
Acetiltransferasas/química , Complejos Multienzimáticos/química , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal/química , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/genética , Acetiltransferasa E N-Terminal/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Especificidad por SustratoRESUMEN
N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.
Asunto(s)
Biomarcadores , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Fenotipo , Adolescente , Adulto , Alelos , Niño , Preescolar , Biología Computacional/métodos , Activación Enzimática , Estabilidad de Enzimas , Facies , Femenino , Sitios Genéticos , Pruebas Genéticas , Genotipo , Humanos , Lactante , Masculino , Modelos Moleculares , Mutación , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/metabolismo , Conformación Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , Adulto JovenRESUMEN
The majority of eukaryotic proteins are N-terminally α-acetylated by N-terminal acetyltransferases (NATs). Acetylation usually occurs co-translationally and defects have severe consequences. Nevertheless, it is unclear how these enzymes act in concert with the translating ribosome. Here, we report the structure of a native ribosome-NatA complex from Saccharomyces cerevisiae. NatA (comprising Naa10, Naa15 and Naa50) displays a unique mode of ribosome interaction by contacting eukaryotic-specific ribosomal RNA expansion segments in three out of four binding patches. Thereby, NatA is dynamically positioned directly underneath the ribosomal exit tunnel to facilitate modification of the emerging nascent peptide chain. Methionine amino peptidases, but not chaperones or signal recognition particle, would be able to bind concomitantly. This work assigns a function to the hitherto enigmatic ribosomal RNA expansion segments and provides mechanistic insights into co-translational protein maturation by N-terminal acetylation.
Asunto(s)
Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Acetilación , Microscopía por Crioelectrón , Humanos , Acetiltransferasa A N-Terminal/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Co-translational N-terminal protein acetylation regulates many protein functions including degradation, folding, interprotein interactions, and targeting. Human NatA (hNatA), one of six conserved metazoan N-terminal acetyltransferases, contains Naa10 catalytic and Naa15 auxiliary subunits, and associates with the intrinsically disordered Huntingtin yeast two-hybrid protein K (HYPK). We report on the crystal structures of hNatA and hNatA/HYPK, and associated biochemical and enzymatic analyses. We demonstrate that hNatA contains unique features: a stabilizing inositol hexaphosphate (IP6) molecule and a metazoan-specific Naa15 domain that mediates high-affinity HYPK binding. We find that HYPK harbors intrinsic hNatA-specific inhibitory activity through a bipartite structure: a ubiquitin-associated domain that binds a hNaa15 metazoan-specific region and an N-terminal loop-helix region that distorts the hNaa10 active site. We show that HYPK binding blocks hNaa50 targeting to hNatA, likely limiting Naa50 ribosome localization in vivo. These studies provide a model for metazoan NAT activity and HYPK regulation of N-terminal acetylation.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/metabolismo , Acetilación , Animales , Sitios de Unión , Cristalografía por Rayos X , Regulación de la Expresión Génica , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Células Sf9 , Especificidad de la EspecieRESUMEN
The NAA10-NAA15 complex (NatA) is an N-terminal acetyltransferase that catalyzes N-terminal acetylation of ~40% of all human proteins. N-terminal acetylation has several different roles in the cell, including altering protein stability and degradation, protein localization and protein-protein interactions. In recent years several X-linked NAA10 variants have been associated with genetic disorders. We have identified a previously undescribed NAA10 c.215T>C p.(Ile72Thr) variant in three boys from two unrelated families with a milder phenotypic spectrum in comparison to most of the previously described patients with NAA10 variants. These boys have development delay, intellectual disability, and cardiac abnormalities as overlapping phenotypes. Functional studies reveal that NAA10 Ile72Thr is destabilized, while binding to NAA15 most likely is intact. Surprisingly, the NatA activity of NAA10 Ile72Thr appears normal while its monomeric activity is decreased. This study further broadens the phenotypic spectrum associated with NAA10 deficiency, and adds to the evidence that genotype-phenotype correlations for NAA10 variants are much more complex than initially anticipated.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Fenotipo , Cardiomiopatía Hipertrófica/patología , Preescolar , Discapacidades del Desarrollo/patología , Estabilidad de Enzimas , Células HeLa , Humanos , Lactante , Discapacidad Intelectual/patología , Masculino , Mutación , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/metabolismo , Unión Proteica , SíndromeRESUMEN
In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification.
Asunto(s)
Proteínas Portadoras/química , Acetiltransferasa A N-Terminal/química , Acetilación , Acetiltransferasas/genética , Dominio Catalítico , Chaetomium , Cristalografía por Rayos X , Humanos , Luz , Modelos Moleculares , Péptidos/química , Unión Proteica , Biosíntesis de Proteínas , Desnaturalización Proteica , Dominios Proteicos , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Dispersión de Radiación , Selenometionina/químicaRESUMEN
N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females.
Asunto(s)
Mutación de Línea Germinal , Discapacidad Intelectual/genética , Mutación Missense , Acetiltransferasa A N-Terminal/deficiencia , Acetiltransferasa E N-Terminal/deficiencia , Acetilación , Femenino , Genes Ligados a X , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Modelos Moleculares , Mosaicismo , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/química , Acetiltransferasa E N-Terminal/genética , LinajeRESUMEN
Structural comparison indicates that the loop region between ß3 and ß4 of SsArd1 is extended relative to the corresponding region in mesophilic Nats, and forms a plastic hydrogen-bond network mainly at two serine residues. Strikingly, two single-point mutants showed â¼3 °C decrease in melting temperature, and two other variants showed â¼7 °C decrease; this correlated with significantly reduced enzymatic activity. To our knowledge, this is the first discovery of a loop region capable of remarkably improving protein thermostability. This provides a novel route to engineer heat-resistant proteins.
Asunto(s)
Proteínas Arqueales/metabolismo , Acetiltransferasa A N-Terminal/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Biocatálisis , Dicroismo Circular , Enlace de Hidrógeno , Mutagénesis Sitio-Dirigida , Acetiltransferasa A N-Terminal/química , Acetiltransferasa A N-Terminal/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sulfolobus solfataricus/metabolismo , Termodinámica , Temperatura de TransiciónRESUMEN
N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish.
Asunto(s)
Acetiltransferasa A N-Terminal/genética , Pez Cebra/embriología , Pez Cebra/genética , Acetilación , Secuencia de Aminoácidos , Animales , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal , Filogenia , Alineación de Secuencia , Especificidad por Sustrato , Pez Cebra/anomalíasRESUMEN
N-terminal acetylation (NTA) is one of the most abundant protein modifications known, and the N-terminal acetyltransferase (NAT) machinery is conserved throughout all Eukarya. Over the past 50 years, the function of NTA has begun to be slowly elucidated, and this includes the modulation of protein-protein interaction, protein-stability, protein function, and protein targeting to specific cellular compartments. Many of these functions have been studied in the context of Naa10/NatA; however, we are only starting to really understand the full complexity of this picture. Roughly, about 40% of all human proteins are substrates of Naa10 and the impact of this modification has only been studied for a few of them. Besides acting as a NAT in the NatA complex, recently other functions have been linked to Naa10, including post-translational NTA, lysine acetylation, and NAT/KAT-independent functions. Also, recent publications have linked mutations in Naa10 to various diseases, emphasizing the importance of Naa10 research in humans. The recent design and synthesis of the first bisubstrate inhibitors that potently and selectively inhibit the NatA/Naa10 complex, monomeric Naa10, and hNaa50 further increases the toolset to analyze Naa10 function.
Asunto(s)
Acetiltransferasa A N-Terminal/fisiología , Acetiltransferasa E N-Terminal/fisiología , Procesamiento Proteico-Postraduccional , Acetilación , Secuencia de Aminoácidos , Animales , Hipoxia de la Célula , Daño del ADN , Regulación de la Expresión Génica , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Humanos , Datos de Secuencia Molecular , Acetiltransferasa A N-Terminal/química , Acetiltransferasa E N-Terminal/química , Neoplasias/enzimología , Estructura Terciaria de ProteínaRESUMEN
Recent studies revealed the power of whole-exome sequencing to identify mutations in sporadic cases with non-syndromic intellectual disability. We now identified de novo missense variants in NAA10 in two unrelated individuals, a boy and a girl, with severe global developmental delay but without any major dysmorphism by trio whole-exome sequencing. Both de novo variants were predicted to be deleterious, and we excluded other variants in this gene. This X-linked gene encodes N-alpha-acetyltransferase 10, the catalytic subunit of the NatA complex involved in multiple cellular processes. A single hypomorphic missense variant p.(Ser37Pro) was previously associated with Ogden syndrome in eight affected males from two different families. This rare disorder is characterized by a highly recognizable phenotype, global developmental delay and results in death during infancy. In an attempt to explain the discrepant phenotype, we used in vitro N-terminal acetylation assays which suggested that the severity of the phenotype correlates with the remaining catalytic activity. The variant in the Ogden syndrome patients exhibited a lower activity than the one seen in the boy with intellectual disability, while the variant in the girl was the most severe exhibiting only residual activity in the acetylation assays used. We propose that N-terminal acetyltransferase deficiency is clinically heterogeneous with the overall catalytic activity determining the phenotypic severity.