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1.
Mol Cell Proteomics ; 12(1): 42-54, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23043182

RESUMEN

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators.


Asunto(s)
Acetiltransferasas/metabolismo , Acetiltransferasas N-Terminal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/genética , Aminoácidos/metabolismo , Línea Celular , Humanos , Lisina/metabolismo , Acetiltransferasas N-Terminal/genética , Procesamiento Proteico-Postraduccional , Proteoma , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia
2.
Am J Hum Genet ; 89(1): 28-43, 2011 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-21700266

RESUMEN

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.


Asunto(s)
Acetiltransferasas/deficiencia , Acetiltransferasas/genética , Cromosomas Humanos X/genética , Genes Ligados a X , Acetilación , Exones , Haplotipos , Humanos , Recién Nacido , Masculino , Mutación , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal , Linaje , Fenotipo
3.
J Biol Chem ; 287(13): 10081-10088, 2012 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-22311970

RESUMEN

N(α)-acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. The NAT hNaa50p preferentially modifies peptides starting with methionine followed by a hydrophobic amino acid. hNaa50p also possesses N(ε)-autoacetylation activity. So far, no eukaryotic NAT has been mechanistically investigated. In this study, we used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. The NMR results, both the substrate binding study and the dynamic data, further indicate that the binding of acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein. This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant.


Asunto(s)
Acetiltransferasas/química , Metionina/química , Modelos Químicos , Péptidos/química , Acetilcoenzima A , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Catálisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Metionina/metabolismo , Mutación , Acetiltransferasa E N-Terminal , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos/genética , Péptidos/metabolismo , Conformación Proteica
4.
Mol Cell Proteomics ; 10(5): M110.004580, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21383206

RESUMEN

The impact of N(α)-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N(α)-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N(α)-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and ß-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and ß-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N(α)-acetyltransferase.


Asunto(s)
Acetiltransferasas/química , Biblioteca de Péptidos , Procesamiento Proteico-Postraduccional , Proteoma/química , Proteínas Recombinantes/química , Acetilación , Acetiltransferasas/biosíntesis , Actinas/química , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Pruebas de Enzimas , Humanos , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal , Polirribosomas/química , Proteínas Recombinantes/biosíntesis , Especificidad por Sustrato
5.
Proc Natl Acad Sci U S A ; 106(20): 8157-62, 2009 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-19420222

RESUMEN

N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys- and Val- N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-Delta nat1-Delta strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-Delta nat1-Delta strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys- termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.


Asunto(s)
Acetiltransferasas/genética , Evolución Molecular , Proteínas Fúngicas/genética , Proteómica/métodos , Acetilación , Arilamina N-Acetiltransferasa/genética , Células HeLa , Humanos , Isoenzimas/genética , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal , Subunidades de Proteína , Especificidad por Sustrato
6.
J Biol Chem ; 284(45): 31122-9, 2009 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-19744929

RESUMEN

Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine N(epsilon)-acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. N(alpha)-terminal acetylation occurs on the ribosome where the alpha amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an N(alpha)-acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.


Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , N-Acetiltransferasa de Aminoácidos/química , N-Acetiltransferasa de Aminoácidos/metabolismo , Acetilación , Acetiltransferasas/genética , Secuencia de Aminoácidos , N-Acetiltransferasa de Aminoácidos/genética , Humanos , Cinética , Datos de Secuencia Molecular , Acetiltransferasa E N-Terminal , Oligopéptidos/química , Oligopéptidos/metabolismo , Alineación de Secuencia , Especificidad por Sustrato
7.
FEBS Lett ; 579(28): 6428-32, 2005 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-16288748

RESUMEN

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1 alpha and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1 alpha and thereby enhances HIF-1 alpha ubiquitination and degradation. Furthermore, ARD1 was proposed to be down-regulated in hypoxia thus facilitating the stabilization of HIF-1 alpha. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1 alpha. However, we find that hARD1 specifically binds HIF-1 alpha, suggesting a putative, still unclear, connection between these proteins.


Asunto(s)
Acetiltransferasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Acetilación , Anaerobiosis , Células Cultivadas , Humanos , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal
8.
Methods Mol Biol ; 981: 95-102, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23381855

RESUMEN

Protein N-terminal acetylation is a widespread modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs). The various NATs and their specific substrate specificities and catalytic mechanisms are far from fully understood. We here describe an in vitro method based on reverse-phase HPLC to quantitatively measure in vitro acetylation of NAT oligopeptide substrates, enabling the determination of NAT specificity as well as kinetic parameters.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Acetiltransferasa E N-Terminal/metabolismo , Oligopéptidos/metabolismo , Acetilación , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato
9.
Mol Cell Biol ; 30(8): 1898-909, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20154145

RESUMEN

The human NatA protein N(alpha)-terminal-acetyltransferase complex is responsible for cotranslational N-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini. The NatA complex is composed of the catalytic subunit hNaa10p (hArd1) and the auxiliary subunit hNaa15p (hNat1/NATH). Using immunoprecipitation coupled with mass spectrometry, we identified endogenous HYPK, a Huntingtin (Htt)-interacting protein, as a novel stable interactor of NatA. HYPK has chaperone-like properties preventing Htt aggregation. HYPK, hNaa10p, and hNaa15p were associated with polysome fractions, indicating a function of HYPK associated with the NatA complex during protein translation. Knockdown of both hNAA10 and hNAA15 decreased HYPK protein levels, possibly indicating that NatA is required for the stability of HYPK. The biological importance of HYPK was evident from HYPK-knockdown HeLa cells displaying apoptosis and cell cycle arrest in the G(0)/G(1) phase. Knockdown of HYPK or hNAA10 resulted in increased aggregation of an Htt-enhanced green fluorescent protein (Htt-EGFP) fusion with expanded polyglutamine stretches, suggesting that both HYPK and NatA prevent Htt aggregation. Furthermore, we demonstrated that HYPK is required for N-terminal acetylation of the known in vivo NatA substrate protein PCNP. Taken together, the data indicate that the physical interaction between HYPK and NatA seems to be of functional importance both for Htt aggregation and for N-terminal acetylation.


Asunto(s)
Acetiltransferasas/metabolismo , Arilamina N-Acetiltransferasa/metabolismo , Proteínas Portadoras/metabolismo , Isoenzimas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Acetiltransferasas/genética , Proteínas Adaptadoras Transductoras de Señales , Arilamina N-Acetiltransferasa/genética , Proteínas Portadoras/genética , Ciclo Celular/fisiología , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Proteína Huntingtina , Isoenzimas/genética , Chaperonas Moleculares/genética , Complejos Multiproteicos/metabolismo , Acetiltransferasa A N-Terminal , Acetiltransferasa E N-Terminal , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
10.
BMC Proc ; 3 Suppl 6: S5, 2009 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-19660098

RESUMEN

Protein acetylation is a common modification that plays a central role in several cellular processes. The most widely used methods to study these modifications are either based on the detection of radioactively acetylated oligopetide products or an enzyme-coupled reaction measuring conversion of the acetyl donor acetyl CoA to the product CoASH. Due to several disadvantages of these methods, we designed a new method to study oligopeptide acetylation. Based on reverse phase HPLC we detect both reaction products in a highly robust and reproducible way. The method reported here is also fully compatible with subsequent product analysis, e.g. by mass spectroscopy. The catalytic subunit, hNaa30p, of the human NatC protein N-acetyltransferase complex was used for N-terminal oligopeptide acetylation. We show that unacetylated and acetylated oligopeptides can be efficiently separated and quantified by the HPLC-based analysis. The method is highly reproducible and enables reliable quantification of both substrates and products. It is therefore well-suited to determine kinetic parameters of acetyltransferases.

11.
Mol Cell Biol ; 29(13): 3569-81, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19398576

RESUMEN

Protein N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Acetiltransferasas/metabolismo , Apoptosis/fisiología , Isoenzimas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factores de Ribosilacion-ADP/genética , Acetiltransferasas/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Acetiltransferasa C N-Terminal , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/genética
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