Dataset Information

Differential N-terminal COFRADIc analyses of Ogden fibroblasts

ABSTRACT: The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase gene. The affected males harbour a Ser37Pro mutation in the gene encoding hNaa10, the catalytic subunit of NatA, themajor human NAT. In order to understand the detrimental impact of hNaa10 Ser37Pro, we performed structural, molecular and cellular investigations. Structural models and molecular dynamics simulations of the human NatA and its Ser37Pro mutant suggest differences in regions involved in catalysis and at the interface between hNaa10 and the auxiliary subunit hNaa15. In agreement, biochemical data demonstrate a reduced catalytic capacity and an impaired NatA complex formation with hNaa10 Ser37Pro. Patient derived Naa10 mutant cells show reduced Nt-acetylation for a subset of NatA/NatE-type substrates compared to wild type Naa10 cells. Ogden syndrome fibroblasts further display abnormal cell proliferation and migration capacity, possibly linked to perturbed Retinoblastoma- and MYLK-pathways, respectively. Differential N-terminal combined fractional diagonal chromatography (COFRADIC) was used to quantify the degree and define the patterns of in vivo protein Nt-acetylation and here used to investigate whether patient cells derived from an affected Ogden male (carrying the Naa10 S37P mutation) displayed altered levels of protein Nt-acetylation at the proteome-wide level.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin, Fibroblast

SUBMITTER: Line Merethe Myklebust

LAB HEAD: Line Merethe Myklebust

PROVIDER: PXD001282 | Pride | 2016-04-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	R17151_1834_WT_S37P_nr1_Nter_28-32-1.RAW	Raw
	R17152_1834_WT_S37P_nr1_Nter_28-32-2.RAW	Raw
	R17153_1834_WT_S37P_nr1_Nter_28-32-3.RAW	Raw
	R17154_1834_WT_S37P_nr1_Nter_28-32-4.RAW	Raw
	R17155_1834_WT_S37P_nr1_Nter_28-32-5.RAW	Raw

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Publications

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

Myklebust Line M LM Van Damme Petra P Støve Svein I SI Dörfel Max J MJ Abboud Angèle A Kalvik Thomas V TV Grauffel Cedric C Jonckheere Veronique V Wu Yiyang Y Swensen Jeffrey J Kaasa Hanna H Liszczak Glen G Marmorstein Ronen R Reuter Nathalie N Lyon Gholson J GJ Gevaert Kris K Arnesen Thomas T

Human molecular genetics 20141208 7

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at t ...[more]

PMID: 25489052

Similar Datasets

Project description:Co-translational N-terminal (Nt-) acetylation of nascent polypeptides is catalyzed by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence is the major factor determining whether or not a given protein is Nt-acetylated. In humans, six different NATs, denoted NatA-NatF, are identified. In the current study we used N-terminal COFRADIC analysis to define the in vivo substrate specificity of Naa50 (Nat5)/NatE as this has long remained elusive. Three yeast strains were generated; a control strain endogenously expressing yNaa50, a deletion strain depleted of yNaa50 and a strain deleted of yNaa50 but ectopically expressing human Naa50. When comparing the Nt-acetylation status of different N-termini in the control strain with the deletion strain, a reduction in Nt-acetylation for several yeast proteins was observed. To our surprise, these substrates were not of the predicted NatE-type substrates, but rather canonical NatA substrates. Further, ectopic expression of hNaa50 mainly resulted in the Nt-acetylation of a selected class of otherwise Nt-free yeast N-termini besides increasing the degree of Nt-acetylation of several other yeast proteins, and as such (partially) complementing those N-termini displaying reduced Nt-acetylation upon yNAA50-deletion. The preferred substrates of hNaa50 were predominantly Met-starting N-termini including Met-Lys-, Met-Val-, Met-Ala-, Met-Tyr-, Met-Phe-, Met-Leu-, Met-Ser- and Met-Thr, and highly overlapped with the previously identified human Naa60/NatF substrate specificity profile. Identification of several hNaa50 substrates with a small amino acid in the second position also revealed a potential interplay between the NATs and methionine aminopeptidases (MetAPs). The initiator Methionine (iMet) is normally cleaved off by MetAPs when the second amino acid is small, but our in vitro data suggest that in contrast to a free iMet, an Nt-acetylated iMet is not hydrolyzed by MetAPs. Thus, Naa50-mediated Nt-acetylation may potentially act as a mechanism to retain the iMet of proteins with a small amino acid at the second position that normally would be hydrolyzed.

Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).