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1.
J Biol Chem ; 297(6): 101363, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34732320

RESUMEN

Huntington's disease (HD) is a neurodegenerative disorder caused by a poly-CAG expansion in the first exon of the HTT gene, resulting in an extended poly-glutamine tract in the N-terminal domain of the Huntingtin (Htt) protein product. Proteolytic fragments of the poly-glutamine-containing N-terminal domain form intranuclear aggregates that are correlated with HD. Post-translational modification of Htt has been shown to alter its function and aggregation properties. However, the effect of N-terminal Htt acetylation has not yet been considered. Here, we developed a bacterial system to produce unmodified or N-terminally acetylated and aggregation-inducible Htt protein. We used this system together with biochemical, biophysical, and imaging studies to confirm that the Htt N-terminus is an in vitro substrate for the NatA N-terminal acetyltransferase and show that N-terminal acetylation promotes aggregation. These studies represent the first link between N-terminal acetylation and the promotion of a neurodegenerative disease and implicates NatA-mediated Htt acetylation as a new potential therapeutic target in HD.


Asunto(s)
Proteína Huntingtina/metabolismo , Agregado de Proteínas , Acetilación , Humanos , Enfermedad de Huntington/genética , Acetiltransferasa A N-Terminal/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional
2.
Hum Mol Genet ; 28(17): 2900-2919, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31127942

RESUMEN

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 Joven
4.
Structure ; 31(2): 166-173.e4, 2023 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-36638802

RESUMEN

N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a large auxiliary subunit that facilitates catalysis and ribosome targeting for co-translational acetylation. NatC is one of the major NATs containing an NAA30 catalytic subunit, but uniquely contains two auxiliary subunits, large NAA35 and small NAA38. Here, we report the cryo-EM structures of human NatC (hNatC) complexes with and without NAA38, together with biochemical studies, to reveal that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide binding loop for optimal catalysis, respectively. We also note important differences in engagement with a stabilizing inositol hexaphosphate molecule between human and yeast NatC. These studies provide new insights for the function and evolution of the NatC complex.


Asunto(s)
Acetiltransferasa C N-Terminal , Proteínas de Saccharomyces cerevisiae , Humanos , Acetilación , Secuencia de Aminoácidos , Acetiltransferasa C N-Terminal/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Biocatálisis
5.
Eur J Hum Genet ; 31(7): 824-833, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37130971

RESUMEN

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex, also including the accessory protein, NAA15. The full spectrum of human genetic variation in this pathway is currently unknown. Here we reveal the genetic landscape of variation in NAA10 and NAA15 in humans. Through a genotype-first approach, one clinician interviewed the parents of 56 individuals with NAA10 variants and 19 individuals with NAA15 variants, which were added to all known cases (N = 106 for NAA10 and N = 66 for NAA15). Although there is clinical overlap between the two syndromes, functional assessment demonstrates that the overall level of functioning for the probands with NAA10 variants is significantly lower than the probands with NAA15 variants. The phenotypic spectrum includes variable levels of intellectual disability, delayed milestones, autism spectrum disorder, craniofacial dysmorphology, cardiac anomalies, seizures, and visual abnormalities (including cortical visual impairment and microphthalmia). One female with the p.Arg83Cys variant and one female with an NAA15 frameshift variant both have microphthalmia. The frameshift variants located toward the C-terminal end of NAA10 have much less impact on overall functioning, whereas the females with the p.Arg83Cys missense in NAA10 have substantial impairment. The overall data are consistent with a phenotypic spectrum for these alleles, involving multiple organ systems, thus revealing the widespread effect of alterations of the NTA pathway in humans.


Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Microftalmía , Humanos , Femenino , Síndrome , Acetiltransferasa E N-Terminal/genética , Acetiltransferasa E N-Terminal/metabolismo , Genotipo , Discapacidad Intelectual/genética , Acetiltransferasa A N-Terminal/genética , Acetiltransferasa A N-Terminal/metabolismo
6.
Structure ; 29(10): 1094-1104.e4, 2021 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-34019809

RESUMEN

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.


Asunto(s)
Proteínas de Schizosaccharomyces pombe/química , Acetilación , Sitios de Unión , Ácido Fítico/química , Ácido Fítico/metabolismo , Unión Proteica , Multimerización de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo
7.
Elife ; 102021 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-34355692

RESUMEN

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.


Asunto(s)
Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Acetilación , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Acetiltransferasa A N-Terminal/deficiencia , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/deficiencia , Acetiltransferasa E N-Terminal/metabolismo
8.
Elife ; 92020 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-32885784

RESUMEN

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (αSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in the pathogenesis of Parkinson's disease and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-αSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for αSyn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.


Asunto(s)
Acetiltransferasa B N-Terminal , alfa-Sinucleína , Acetilación , Coenzima A/química , Coenzima A/metabolismo , Humanos , Modelos Moleculares , Acetiltransferasa B N-Terminal/antagonistas & inhibidores , Acetiltransferasa B N-Terminal/química , Acetiltransferasa B N-Terminal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
9.
Sci Rep ; 10(1): 15788, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32978498

RESUMEN

Mature frataxin is essential for the assembly of iron-sulfur cluster proteins including a number of mitochondrial enzymes. Reduced levels of mature frataxin (81-20) in human subjects caused by the genetic disease Friedreich's ataxia results in decreased mitochondrial function, neurodegeneration, and cardiomyopathy. Numerous studies of mitochondrial dysfunction have been conducted using mouse models of frataxin deficiency. However, mouse frataxin that is reduced in these models, is assumed to be mature frataxin (78-207) by analogy with human mature frataxin (81-210). Using immunoaffinity purification coupled with liquid chromatography-high resolution tandem mass spectrometry, we have discovered that mature frataxin in mouse heart (77%), brain (86%), and liver (47%) is predominantly a 129-amino acid truncated mature frataxin (79-207) in which the N-terminal lysine residue has been lost. Mature mouse frataxin (78-207) only contributes 7-15% to the total frataxin protein present in mouse tissues. We have also found that truncated mature frataxin (79-207) is present primarily in the cytosol of mouse liver; whereas, frataxin (78-207) is primarily present in the mitochondria. These findings, which provide support for the role of extra-mitochondrial frataxin in the etiology of Friedreich's ataxia, also have important implications for studies of mitochondrial dysfunction conducted in mouse models of frataxin deficiency.


Asunto(s)
Modelos Animales de Enfermedad , Ataxia de Friedreich/patología , Proteínas de Unión a Hierro/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Corazón/fisiología , Humanos , Proteínas de Unión a Hierro/genética , Hígado/metabolismo , Ratones , Mitocondrias/genética , Frataxina
10.
Methods Enzymol ; 626: 271-299, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31606079

RESUMEN

N-terminal acetylation is a co- and post-translational modification catalyzed by the conserved N-terminal acetyltransferase (NAT) family of enzymes. A majority of the human proteome is modified by the human NATs (NatA-F and H), which are minimally composed of a catalytic subunit and as many as two auxiliary subunits. Together, NATs specifically regulate many cellular functions by influencing protein activities such as their degradation, membrane targeting, and protein-protein interactions. This chapter will describe methods developed for their preparation, and their biochemical and structural characterization. This will include methodologies for expression and purification of recombinant NAT protein, kinetic assays, biochemical and biophysical assays, and strategies for structural studies.


Asunto(s)
Acetiltransferasas N-Terminal/química , Acetiltransferasas N-Terminal/metabolismo , Animales , Dominio Catalítico , Línea Celular , Clonación Molecular/métodos , Cristalización/métodos , Cristalografía por Rayos X/métodos , Pruebas de Enzimas/métodos , Escherichia coli/genética , Humanos , Modelos Moleculares , Acetiltransferasas N-Terminal/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulación hacia Arriba
11.
Structure ; 26(7): 925-935.e8, 2018 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-29754825

RESUMEN

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 Especie
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