Knockdown of Human N -Terminal Acetyltransferase Complex C Leads to p53-Dependent Apoptosis and Aberrant Human Arl8b Localization

Article (PDF Available)inMolecular and Cellular Biology 29(13):3569-81 · May 2009with46 Reads
DOI: 10.1128/MCB.01909-08 · Source: PubMed
Protein Nα-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.
MOLECULAR AND CELLULAR BIOLOGY, July 2009, p. 3569–3581 Vol. 29, No. 13
0270-7306/09/$08.000 doi:10.1128/MCB.01909-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Knockdown of Human N
-Terminal Acetyltransferase Complex C
Leads to p53-Dependent Apoptosis and Aberrant Human
Arl8b Localization
Kristian K. Starheim,
Darina Gromyko,
Rune Evjenth,
Anita Ryningen,
Jan Erik Varhaug,
Johan R. Lillehaug,
and Thomas Arnesen
Department of Molecular Biology, University of Bergen, N-5020 Bergen, Norway
; Department of Surgical Sciences, University of
Bergen, N-5020 Bergen, Norway
; Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway
; and
Department of Medicine, Haukeland University Hospital, N-5021 Bergen, Norway
Received 18 December 2008/Returned for modification 19 February 2009/Accepted 18 April 2009
Protein N
-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.
Approximately 50% of yeast proteins and 80% of mamma-
lian proteins are N-terminally acetylated (9). The impact of
this modification on a large range of cellular processes, includ-
ing links to cancer development, is being increasingly recog-
nized. However, knowledge about the functional importance
and underlying mechanisms of protein N
-terminal acetylation
is still limited.
In yeast, three NAT complexes, NatA, NatB, and NatC,
account for most of the protein N
-terminal acetylation in
what is believed to be a cotranslational process. They acetylate
different subsets of proteins as defined by the sequence and
nature of the N-terminal amino acid residues of the substrates
(18, 28, 29).
Yeast NatC contains the catalytic subunit Mak3p and the
auxiliary subunits Mak10p and Mak31p, where all three sub-
units are necessary for NatC activity. The NatC complex po-
tentially acetylates methionine N termini when the second
residue is one of the hydrophobic amino acids leucine, isoleu-
cine, phenylalanine, or tryptophan. The mutant yeast strains
mak3-, mak10-, and mak31- display similar phenotypes:
lack of acetylation of substrates in vivo, diminished growth at
37°C in medium without fermentable carbon sources (30), de-
fective L-A virus propagation (39), and loss of telomere elon-
gation (11). In a screen for conserved synthetic lethal genetic
interactions in Saccharomyces cerevisiae and Schizosaccharo-
myces pombe, MAK10 was found to be part of a core genetic
interaction network. In this screen, NatC-mediated acetylation
was suggested to be critical for efficient DNA replication in
eukaryotes (15). Deletion of MAK10 has also been observed to
increase sensitivity to rapamycin (41), indicating a link to the
mTOR pathway. Identified NatC substrates include the Gag
major coat protein of the L-A virus (35), the tRNA methyl-
transferase Trm1p-II (25), and the ARF-like GTPase Arl3p
(12, 32). For all these substrates, NatC-mediated acetylation is
necessary for normal protein activity. In the case of Arl3p,
NatC-mediated N-terminal acetylation is important for its
Golgi apparatus association (12, 32).
In humans, the NatA and NatB complexes have been iden-
tified. The human NatA complex (hNatA) is composed of the
hNat1/NATH, hArd1, and hNat5/hSan subunits (2, 3). Ard1,
the catalytic subunit of NatA, is essential for growth of Cae-
norhabditis elegans (33), the parasite Trypanosoma brucei (21),
and human cells (6, 16). Human Ard1 has been suggested as a
potential cancer drug target (6, 8). The hNatB complex con-
sists of the subunits hNat3 and hMdm20. Knockdown of the
hNatB subunits disturbs cell proliferation and cell cycle pro-
gression, and hNat3 has been linked to tumor growth (1, 34). A
vertebrate NatC complex has been identified, consisting of
Mak3, Mak31, and the Mak10p homologue embryonic growth-
associated factor. Knockdown of zebrafish embryonic growth-
associated factor induced embryonic growth lethality and re-
duced the protein level and signaling activity of zTOR protein
kinase (38).
In the present communication, we describe the hNatC com-
plex. It is evolutionarily conserved from yeast with respect to
subunit composition, localization, enzymatic activity, and sub-
strate specificity. Knockdown of individual hNatC subunits
leads to cell growth defects and p53-dependent apoptosis.
* Corresponding author. Mailing address: Department of Molecular
Biology, University of Bergen, Thormøhlensgate 55, N-5020 Bergen,
Norway. Phone: 47 55584539. Fax: 47 55589683. E-mail: Thomas
Published ahead of print on 27 April 2009.
Knockdown of hMAK3 induces a change in the subcellular
localization of hArl8b, suggesting that hNatC-mediated acety-
lation is necessary for hArl8b localization. Our present data
suggest an important role for hNatC-mediated acetylation in
various cellular processes necessary for normal cell viability in
Construction of plasmids. Plasmids encoding Xpress and/or V5-tagged
hMak10, hMak3 (Nat12), hMak31 (Lsmd1), and Lsm8 were constructed from
cDNA made from total RNA isolated from human HEK293 and HeLa cells.
PCR products were inserted into the TOPO TA vectors pcDNA 3.1/V5-His
TOPO and pcDNA4/HisMax-TOPO (Invitrogen) according to the instruction
manual. SuperScript II reverse transcriptase (Invitrogen) was used to make
cDNA. Plasmid encoding Xpress-tagged hNat5 was previously described (3).
Plasmid encoding MBP-hMak3 was constructed by subcloning hMak3 from pX-
press-hMak3 to the pETM-41 vector. Primer sequence information is available
upon request. pETM-41 was generously provided by G. Stier, EMBL, Heidel-
berg, Germany. hARL8b-GFP plasmid was kindly provided by S. Munro, MRC
Laboratory of Molecular Biology, Cambridge, United Kingdom.
Cell culture and transfection. HeLa cells and HEK293 cells were cultured and
transfected as described previously (2). CAL-62 cells (human thyroid anaplastic
carcinoma; DSMZ no. ACC 507) were cultured in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum. HCT116 (human colon cancer carcinoma)
and parental p53
cells were kindly provided by F. Bunz, B. Vo
gelstein, and K. W. Kinzler (Johns Hopkins University School of Medicine and
Howard Hughes Medical Institute, MD) and were routinely maintained in
McCoy’s 5A medium supplemented with 10% normal calf serum. To specifically
inhibit caspase activities 20 M ZVAD-fmk (R&D Systems Europe Ltd.) was
added when appropriate. Small interfering RNA (siRNA)-mediated knockdown
was performed using Dharmafect 1 transfection reagent (Dharmacon) according
to the instruction manual. Gene-specific SMART pool siRNAs were purchased
from Dharmacon and used at a final concentration of 20 to 50 nM to silence the
hMAK3 (NAT12), hMAK10, and hMAK31 (LSMD1) genes (sihMAK3 [NAT12],
catalog no. M-009961-02; sihMAK10, catalog no. L-021268-01; sihMAK31
[LSMD1], catalog no. L-014876-01). Nontargeting pool (catalog no. D-001810-
10) and ON-TARGETplus GAPDH control pool (catalog no. D-001830) were
used as controls. Where indicated, two individual siRNAs from the sihMAK3
SMART pool were used for knockdown of hMAK3 (Dharmacon catalog nos.
D-009961-01 and D-009961-05).
Immunoprecipitation. HeLa and HEK293 cells were harvested and lysed as
described previously (2). The cell lysates were incubated for2hat4°Cwith
specific antibody (2 g) before 50 l of protein A/G-agarose was added. After
incubation for 16 h, centrifugation, and washing three times in 2 phosphate-
buffered saline, the samples were subjected to sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and Western blotting.
Isolation of polysomes. Total ribosome isolation was performed using a mod-
ification of previously described methods (27, 37). Approximately 2 10
HEK293 cells were used per experiment. Prior to harvesting, cells were treated
with 10 g/ml cycloheximide for 5 min at 37°C. Cells were harvested and lysed
with KCl ribosome lysis buffer (1.1% [wt/vol] KCl, 0.15% [wt/vol] triethano-
lamine, 0.1% [wt/vol] magnesium acetate, 8.6% [wt/vol] sucrose, 0.05% [wt/vol]
Na-deoxycholate, 0.5% [vol/vol] Triton X-100, 0.25% [vol/vol] Pefabloc), and
incubated on ice for 15 min. After removing nuclei and membranes by centrif-
ugation at 400 g for 10 min, 700 l cell lysate was centrifuged at 436,000 g
for 25 min on a 0.4-ml cushion of 25% sucrose in KCl ribosome lysis buffer using
an MLA-130 rotor (Beckman, Geneva, Switzerland). Pellets were resuspended in
ribosome lysis buffer with the indicated KCl concentrations, followed by ultra-
centrifugation as described above. Pellets were resuspended in KCl ribosome
lysis buffer and prepared for analysis by SDS-PAGE and Western blotting.
Immunofluorescence. HeLa cells grown on coverslips were washed in phos-
phate-buffered saline, fixed in methanol, permeabilized in 0.1% Triton X-100,
and blocked in 10% bovine serum albumin (BSA). Primary antibodies were
anti-V5 or anti-Xpress (Invitrogen). Secondary antibodies were Alexa Fluor
488-, Alexa Fluor 594-, or Texas Red-conjugated immunoglobulin G (Invitro-
gen). Blue Hoechst 33342 or 4,6-diamidino-2-phenylindole staining was used to
stain the nucleus.
For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end la-
beling (TUNEL) assays, cells were incubated in TUNEL reaction mixture (In
Situ Cell Death Detection kit with TMR red; Roche) for1hat37°C. Positive and
negative controls for each sample were obtained by treating samples with DNase
I or incubation in TUNEL label reaction mixture without enzyme, respectively.
For investigation of hArl8b localization after hMAK3 knockdown, hMAK3
knockdown cells were transfected with hARL8b-GFP. Cells were treated with
ZVAD (20 M) after knockdown of hMAK3 to prevent induction of apoptosis.
Anti-LAMP-1 (Santa Cruz Biotechnology) was used as a lysosomal marker. The
microscopic recordings were processed by deconvolution (Leica 4000 software).
The data for quantification of hArl8b-green fluorescent protein (GFP) localiza-
tion after hMAK3 knockdown are shown as the means of at least 500 cells
counted in five to six samples from three independent experiments.
Western blotting. SDS-PAGE and Western blotting were performed as de-
scribed previously (5). The following antibodies were used: anti-V5, anti-Xpress,
and anti-HA (Invitrogen); anti-L26 (Novus Biologicals); anti--tubulin and anti-
Mdm2 clone SMP14 (Sigma); anti-cytochrome c (Santa Cruz Biotechnology);
anti-cleaved -Fodrin (Asp1185), anti-cleaved poly(ADP) ribose polymerase
(PARP; Asp214), anti-p53, anti-phospho-p53 (Ser15), and anti-phospho-p53
(Ser37) (Cell Signaling Technology). Anti-hMak3 (Biogenes) was generated by
immunizing rabbits with purified hMak3 protein produced in Escherichia coli,
followed by immunoglobulin G isolation from the resulting sera. Horseradish
peroxidase-linked anti-mouse and anti-rabbit antibodies were from Amersham
Biosciensce (Little Chalfont, Buckinghamshire, United Kingdom).
In vitro N
-acetyltransferase assays. Expression and purification of maltose
binding protein (MBP)-hMak3 was carried out as previously described (7).
Acetylation assays were performed with 30 l MBP-hMak3 (2.78 M stock), 5 l
synthetic peptide (1 mM stock of custom-made peptides from Biogenes),
5 l[
C]acetyl coenzyme A (50 Ci, 2.07 GBq/mmol; GE Healthcare), and
250 l acetylation buffer (50 mM Tris-HCl, pH 8.5, 1 mM dithiothreitol, 800 M
EDTA, 10 mM Na-butyrate, 10% glycerol). The mixture was incubated for2hat
37°C with rotation and then added to 250 l SP Sepharose (50% slurry in 0.5 M
acetic acid; Sigma) and incubated on a rotor for 5 min. The mixture was centri-
fuged and the pellet was washed three times with 0.5 M acetic acid and one time
in methanol. Radioactivity in the peptide-containing pellet was determined by
scintillation counting. All custom-made peptides contain seven unique amino
acids from the N terminus, since these residues are the major determinants for
-terminal acetyltransferase specificity. The next 17 amino acids are identical to
the ACTH peptide sequence to maintain a positive charge facilitating peptide
solubility and effective isolation by cation exchange Sepharose beads. The
ACTH-derived lysines were replaced by arginines to eliminate any potential
interference from N
acetylation. The peptide sequences were as follows: hArl8b
RRRRPVRVYP[OH]; high-mobility group protein A1 (P17096), [H]SESSSKSR
Semiquantitative and real-time PCR. Verification of gene-specific silencing
after siRNA transfection was done by semiquantitative real-time PCR (RT-
PCR) using specific primers (primer sequence information is available upon
request). Relative gene expression levels of p53-induced genes in HeLa cells
transfected with siRNA against hMAK3 were determined by real-time quantita-
tive PCR. The sequences of the primers used to amplify the fragments of NOXA,
KILLER/DR5, and FAS cDNA are available upon request. The primers for
amplifying fragments of hMAK3 were designed to target the acetyltransferase
domain. We used RPLP2 (ribosomal protein large P2) primers to amplify ref-
erence gene. Templates (equal amounts of cDNA) and primers were mixed with
components from the LightCycler 480 SYBR green I Master Mix kit (Roche
Applied Sciences). Reactions in triplicates were carried out in the LightCycler
480 real-time PCR machine (Roche Applied Science) under the following con-
ditions: initial denaturation at 95°C for 5 min and then 40 cycles of denaturation
at 95°C for 10 s, annealing at 54°C for 10 s, and extension at 72°C for 10 s.
Melting curves were obtained to examine the purity of amplified products. CP
and concentrations of amplicons were calculated with LightCycler 480 software
v.1.5.0 SP1 using the second derivative max method. Normalization of obtained
data was done as follows: {(concentration of gene [NOXA, KILLER/DR5, FAS,
or hMAK3] in a sample)/(concentration of reference gene RPLP2 in a sample)}/
{(concentration of gene [NOXA, KILLER/DR5, FAS,orhMAK3] in control)/
(concentration of reference gene RPLP2 in the control)}. P values were obtained
by independent t test.
Cell cycle analysis. After harvesting of approximately 1 10
cells by trypsin-
EDTA treatment, the cells were processed by fluorescence-activated cell sorting
analysis as described in previous studies (6).
BrdU incorporation assay. Cells growing in a 96-well plate were transfected
with correspondent siRNAs. After 48 h the proliferation rate was determined by
measuring the amount of bromodeoxyuridine (BrdU) incorporated into nuclear
DNA using the bromodeoxyuridine incorporation assay (Cell Proliferation
ELISA, BrdU, chemiluminescent; Roche Applied Science, Mannheim, Ger-
many) according to the instructions of the manufacturer.
WST assay. HeLa cells were seeded at 3 10
cells per well in 96-well plates
and transfected with 50 nM correspondent siRNAs after 16 h. After 72 h water-
soluble tetrazolium 1 (WST-1; Roche) was added to each well (1:10) for1hand
absorbance at 450 nm was determined. Four replicates were used for each
sample. Reduction in cell viability was expressed as the ratio of absorbance
(sample) versus absorbance (control) multiplied by 100%.
Identification of human NatC complex components. In the
search for human homologues of the yeast NatC components
Mak3p, Mak10p, and Mak31p, we used the protein sequences
of these as query sequences in BLAST searches against the
human proteome. We identified the putative genes encoding
human homologues of Mak3p (NAT12; GeneID 122830),
Mak10p (MAK10; GeneID 60560), and Mak31p (LSM8;
GeneID 51691). Previously, Wenzlau and coworkers presented
FIG. 1. Alignment of Mak3p and Mak31p homologues. Identities are given in dark gray. Conservative substitutions are given in light gray.
(A) Putative homologues of S. cerevisiae (Sc) Mak3p: human (Hs) Nat12, Mus musculus (Mm) Nat12, S. pombe (Sp) NP_596246, D. melanogaster
(Dm) NP_181348, A. thaliana (At) NP_181348, C. elegans (Ce) NP_504411. (B) Putative homologues of yeast Mak31p: Hs Lsm8, Hs Lsmd1, Sp
NP588509, Dm NP_647660, M. sativa (Ms) P24715, At NP_187757, Thermoplasma acidophilum (Ta) NP_394385, Ce NP_498708. The alignments
were performed using JalView (14) and MAFFT sequence aligner (22).
a vertebrate NatC complex of Mak10, Mak3, and Mak31 pro-
teins (38). They suggested that hMAK31 is LSMD1 (GeneID
84316) and hMAK3 is NAT13/hNAT5 (GeneID 80218). How-
ever, hNat5 is the human homologue of yeast Nat5p and the
Drosophila melanogaster San, and it was previously identified as
the third subunit of the NatA complex (3, 18, 40). Both hNat12
and hNat13/hNat5 are predicted acetyltransferases of the GNAT
superfamily. Pair-wise BLAST analysis between the different
human candidates and the yeast proteins demonstrated that
yeast Mak3p was more similar to hNat12 than to hNat13/
hNat5/hSan (data available upon request) and that Lsm8 was
more similar to yeast Mak31p than was Lsmd1 (data available
upon request). Figure 1 displays alignments of putative Mak3
and Mak31 candidates from different species.
To determine which of these proteins interacts to form the
human NatC complex, the open reading frames encoding the
candidate proteins were amplified from HeLa cell cDNA and
used to construct plasmids expressing the candidate genes as
Xpress- and/or V5-tagged proteins. For hMAK10, a splice
event had taken place on the candidate retained from the
PCR, resulting in the loss of nucleotides 1465 to 1704 com-
pared to the predicted sequence (data available upon request).
As this was the only observed product, possibly representing a
biological feature of hMAK10 expression in HeLa cells, the
product was further tested as an hNatC subunit.
We tested all components for their ability to associate with
hMak10, since the sequence information identified hMak10 as
the most likely hNatC subunit compared to the hMak3 and
hMak31 candidates. The results demonstrated that hMak10
interacted with hNat12 and Lsmd1. We were not able to detect
FIG. 2. hMak10, hNat12/hMak3, and Lsmd1/hMak31 interact to
form a stable complex. (A) HeLa cells expressing hMak10-V5 and
Xpress-lacZ (negative control), Xpress-hNat12, Xpress-hNat5,
Xpress-Lsm8, or Xpress-Lsmd1 were harvested and the lysates were
immunoprecipitated (IP) with anti-Xpress antibody (Invitrogen). Im-
munoprecipitated proteins were identified by Western blotting using
the indicated antibodies. (B) Cells expressing hMak10-V5, Lsmd1-V5,
and Xpress-hNat12 or Xpress-lacZ (negative control) were immuno-
precipitated as for panel A using an anti-Xpress antibody in the im-
munoprecipitation. Molecular mass (in kDa) is indicated on the left-
hand side. Results shown are representative of more than three
independent experiments.
FIG. 3. Subcellular localization of hMak10-V5, Xpress-hMak3, and
Xpress-hMak31. HeLa cells were transfected with hMak10-V5 (A and
B), Xpress-hMak3 (C and D), or Xpress-hMak31 (E and F). Anti-V5
antibodies and Alexa-594-conjugated anti-mouse antibodies were used
to visualize hMak10-V5 (A). Anti-Xpress antibodies and Alexa-488-
conjugated anti-mouse antibodies were used to visualize Xpress-
hMak3 (C). Anti-Xpress antibodies and Texas Red-conjugated anti-
mouse antibodies were used to visualize Xpress-hMak31 (E). 4,6-
diamidino-2-phenylindole staining was used to visualize the nuclei of
the cells (B, D, and F). Bar, 25 m.
any interaction between hMak10 and hNat5 or Lsm8 (Fig. 2A).
It was technically difficult to detect the Xpress-Lsm8 protein by
Western blotting, but the significant expression of Xpress-
Lsm8 in HeLa cells was confirmed by immunofluorescence
microscopy (data not shown). To confirm that hMak10, hNat12,
and Lsmd1 associated to form a complex, we expressed
Xpress-hNat12, hMak10-V5, and Lsmd1-V5 in HEK293-cells
and used an Xpress antibody for immunoprecipitation. When
FIG. 4. hNatC subunits cosediment with polysomal fractions in a salt-sensitive manner. (A) Polysomal pellets from HeLa cells expressing hMak10-V5,
Xpress-hMak3, and Xpress-hMak3 were resuspended in buffer containing increasing concentrations of KCl. Cell lysate (L), supernatant post-first
ultracentrifugation (S), and polysomal pellets after KCl treatment were analyzed by SDS-PAGE and Western blotting. The membrane was incubated with
anti-V5, anti-Xpress, anti-L26 (ribosomal protein), and anti-CytC antibodies. Molecular mass markers (in kDa) are indicated on the left-hand side.
(B) For endogenous hMak3, untransfected cells were treated as described for panel A and analyzed with anti-hMak3 antibody. Molecular mass (in kDa)
is indicated on the left-hand side. Results shown are representative of more than three independent experiments.
FIG. 5. hMak3 displays sequence-specific N-acetyltransferase activity. E. coli-expressed and purified MBP-hMak3 was analyzed for N
acetyltransferase activity using peptides differing in their N-terminal residues and [1-
C]acetyl coenzyme A. The acetyl incorporation was
determined by isolation of the peptides followed by scintillation counting. Experiments were performed three to five times for each peptide. Error
bars indicate standard deviations.
analyzing the complexes by SDS-PAGE and Western blotting
with anti-V5, specific signals were detected for hMak10-V5
and Lsmd1-V5 compared to the control (Fig. 2B). The inter-
action between hNat12 and Lsmd1 was further confirmed by
expressing Lsmd1-V5 in HeLa cells, using a hNat12 antibody
for immunoprecipitation. When analyzing the complexes by
SDS-PAGE and Western blotting as described above, specific
signals for Lsmd1-V5 were detected (data not shown). Due to
technical difficulties we were not able to use the hNat12-spe-
cific antibody for analyzing the interaction between hNat12
and hMak10. Based on these results, we propose that hNat12
is the hMak3, while Lsmd1 is the hMak31. The names hMak3
and hMak31 will be used in the following discussion of our
The human NatC subunits are located in the cytoplasm and
associated with ribosomes. To determine the subcellular local-
ization of the hNatC components, HeLa cells were used for
immunostaining analysis. hMak3 and hMak10 localized almost
exclusively to the cytoplasm while hMak31 also localized to the
nucleus (Fig. 3).
The NAT complexes are believed to cotranslationally acet-
ylate nascent polypeptides in yeast, and it has been shown that
the NatA complex associates with ribosomes via its Nat1p
subunit (18). Similarly, the components hNat1 and hArd1 of
hNatA form a complex cosedimenting with ribosomes (2). To
investigate the ribosome-binding properties of the human
NatC complex, we isolated polysomes from HeLa cells over-
expressing the different hNatC subunits. Western blot analysis
demonstrated that xp-hMak3, hMak10-V5, and hMak31-V5
cosediment with ribosomal pellets, suggesting that all subunits
are stably associated with ribosomes (Fig. 4A). Endogenous
hMak3 also cosediments with ribosomal pellets in a salt-sen-
sitive manner (Fig. 4B). These results support the hypothesis
that hMak3, hMak10, and hMak31, as a complex, are involved
in cotranslational acetylation. In addition, the hNatC subunits
were present in the nonribosomal fraction. Thus, they may be
dynamically associated with the ribosomes, or they may have
functions that are independent of cotranslational acetylation.
Interestingly, there seem to be some differences in the amount
of bound and unbound forms of the various subunits.
Human Mak3 displays N-terminal acetyltransferase activ-
ity. The yeast NatC complex acetylates Met-Leu/Ile/Phe/Trp N
termini. To determine whether the human NatC complex ex-
presses a similar activity, the catalytic subunit hMak3 was ex-
pressed with an MBP tag in E. coli and purified using several
steps of His affinity column and gel filtration. The purified
MBP-hMak3 was assayed for in vitro activity using oligopep-
tides with various N-terminal amino acid residues as sub-
strates. Significant activity was observed for peptide substrates
having the N termini MLALI, MLGTG, and MLGTE. Also,
some activity was obtained toward the peptide starting with
MLGPE (Fig. 5). The predicted NatA and NatB substrates,
SESSS and MDELF, respectively, were not significantly acety-
lated. Thus, it seems that hMak3 is the catalytic subunit of
hNatC and that its substrate specificity is highly conserved
from yeast. Interestingly, it is clear from these data that the
substrate specificity of NatC at least in part is contained within
the catalytic subunit alone.
NatC knockdown induces p53-dependent cell death and
growth arrest in human cell lines. To investigate the biological
FIG. 6. hMAK3,hMAK10,orhMAK31 knockdown induces apoptosis in HeLa cells. (A) Cells cultured in six-well plates were transfected with
50 nM hMAK3,hMAK10,orhMAK31 SMART pool siRNAs or control siRNA (siGAPDH or nontargeting siRNA). After 48 h, total RNA was
isolated and processed by RT-PCR with specific primers against the genes of interest. (B) At 48 h posttransfection the cell proliferation rate was
determined using a BrdU assay, measuring the amount of bromodeoxyuridine incorporated into nuclear DNA. The results are given as percent
mitogenic activity. Error bars (B and C) represent standard deviations. P values for independent t tests for samples versus control are indicated
, P 0.001). (C) At 72 h posttransfection the cell viability was measured using a WST assay. The results are given as percent cell viability. P
values for independent t tests for samples versus control are indicated (
, P 0.00001). (D) Live cell imaging of hMAK3 knockdown cells 72 h
posttransfection. Arrowheads indicate apoptotic cells. Hoechst 33342 staining was used to stain the nuclei. Phase contrast (PH) was used to
visualize cells. (E) PARP cleavage was observed by harvesting cells 72 h posttransfection and analyzing cell lysates by Western blotting. The
membrane was incubated in anti-cleaved PARP and anti--tubulin (loading control). All experiments were performed a minimum of three times.
(F) At 72 h posttransfection hMAK3,hMAK10,orhMAK31 knockdown cells were analyzed for DNA breaks by using a TUNEL assay. Blue Hoechst
33342 staining was used to visualize the nuclei. Cells were visualized by phase contrast (PH).
FIG. 7. Cell cycle analysis of hMAK3,hMAK10, and hMAK31
knockdown cells. Flow cytometric cell cycle analysis results are shown
for hMAK3,hMAK10, and hMAK31 knockdown cells at 72 h posttrans-
fection. Experiments were performed three times, and representative
values are given.
importance of the human NatC complex, we performed
siRNA-mediated knockdown of the genes encoding the hNatC
subunits hMak10, hMak3, and hMak31. RT-PCR demon-
strated efficient knockdown of respective RNA transcripts in
HeLa cells (Fig. 6A). Using a BrdU assay we detected a sig-
nificantly diminished rate of DNA synthesis in cell cultures
treated with siRNAs targeting hMAK3,hMAK10,orhMAK31
(Fig. 6B). The WST-1 cell proliferation/viability assay clearly
demonstrated that knockdown of all three hNatC subunits
reduced cell viability (Fig. 6C). By live cell microscopy, we also
observed cell death after treatment with siRNAs targeting
hMAK3 (Fig. 6D). To investigate whether caspases were acti-
vated as a consequence of a decrease in the hNatC complex
level, we assayed for cleavage of the known caspase target
PARP. Western blot analysis demonstrated that knockdown of
all three hNatC subunits induced PARP cleavage (Fig. 6E). A
TUNEL assay confirmed the presence of DNA breaks as a
further indication of apoptosis (Fig. 6F).
Furthermore, we performed fluorescence-activated cell sort-
ing cell cycle analysis on hNatC knockdown cells. Here we
observed an increase in the sub-G
fraction, representing
nuclear fragmentation (Fig. 7). Knockdown of hMAK3,
hMAK10,orhMAK31 in the colon carcinoma cells HCT116
) induced apoptosis (Fig. 7, top panel). However, in
similar experiments using HCT116 (p53
) cells, apoptosis
was not observed, indicating that p53 is essential for the hNatC
knockdown-mediated induction of apoptosis (Fig. 7, middle
panel). Accumulation of hNatC knockdown cells in the sub-
fraction was also observed for HeLa cells (Fig. 7, lower
panel). Adding the pan-caspase inhibitor ZVAD reversed the
accumulation, supporting that sihNatC-treated cells
undergo caspase-mediated cell death (data not shown).
Western blot assays of HeLa cell extracts 72 h posttransfec-
tion demonstrated that hMAK3 knockdown by two different
hMAK3-specific siRNAs induced a decrease in Mdm2 levels
(Fig. 8A) and a stabilization of p53. The Mdm2 reduction may
FIG. 8. hMAK3 knockdown induces apoptosis via p53 stabilization and transcriptional activation. (A) HeLa cells cultured in six-well plates were
transfected with 50 nM hMAK3-1 or hMAK3-2 taken from the SMART pool sihMAK3 or with control siRNA (nontargeting siRNA). Daunorubicin
(Dau) treatment was used as a positive control for apoptosis. At 72 h posttransfection, cell lysates were analyzed by Western blotting. The
membrane was incubated with anti-cleaved -Fodrin (Asp1185), anti-cleaved PARP (Asp214), anti-Mdm2, anti-p53, anti-phospho-p53 (Ser15),
anti-phospho-p53 (Ser37), anti-hMak3, and anti--tubulin (loading control). Experiments were performed a minimum of three times, and
representative results are shown. (B) Cells were treated as for panel A, but after harvesting, total RNA was isolated and processed by quantitative
RT-PCR with gene-specific primers against hMAK3, KILLER/DR5, FAS, and NOXA. Error bars indicate standard deviations. P values for
independent t tests for samples versus control are indicated (
, P 0.0001). Values indicated in the diagram are given in the table below.
potentially contribute to the stabilization of p53. Previously, it
was demonstrated that phosphorylation of p53 at Ser15 and
Ser37 mediates transcriptional activation of p53 (23). The ob-
servation that p53 is phosphorylated at Ser37 in hMAK3 knock-
down cells points to a possible pathway through which p53
stabilization and/or activation is affected (Fig. 8A). In contrast
to daunorubicin-treated cells, Ser15 of p53 was not phosphor-
ylated in sihMAK3-treated cells (Fig. 8A). Apoptosis was con-
firmed by the detection of cleaved PARP and -Fodrin. Using
anti-hMak3, we confirmed efficient hMAK3 knockdown at the
endogenous protein level, and furthermore we demonstrated
that daunorubicin-induced apoptosis caused significantly re-
duced levels of hMak3 protein (Fig. 8A). Daunorubicin treat-
ment did not alter the mRNA levels of hMAK3 (Fig. 8B),
suggesting that the daunorubicin-induced reduction of hMak3
protein occurs directly at the protein level. To further elabo-
FIG. 9. Expression of exogenous hMak3 rescues hMAK3 knock-
down phenotypes. (A) The viability of hMAK3 knockdown cells ex-
pressing exogenous hMak3-V5 was compared to hMAK3 knockdown
cells not expressing exogenous hMak3. Cell viability was measured 72 h
posttransfection using a WST-1 assay. Results are given as percent cell
viability. Experiments were performed in two parallel assays using two
individual hMAK3 siRNAs. DharmaFECT Duo transfection reagent
was used for cotransfection. Nontargeting SmartPool siRNA and
lacZ-V5 were used as cotransfection controls. HeLa cells were used in
all experiments. P values for hMak3 knockdown rescued with exoge-
nous expression of hMak3 obtained with paired t tests are indicated:
P 0.004;
, P 0.001. (B) Cells were treated as for panel A, and
cell lysates were analyzed by SDS-PAGE and Western blotting. The
membrane was incubated with anti-cleaved PARP (Asp214), anti-p53,
anti-phospho-p53 (Ser37), anti-hMak3, anti-V5, and anti--tubulin
(loading control). Results shown are representative of three indepen-
dent experiments.
FIG. 10. hMAK3 knockdown alters hArl8b localization. hArl8b-GFP
was transfected into siControl cells (A to D) and sihMAK3 cells (E to H).
(A and E) Localization of hArl8b-GFP (green); (B and F) the lysosomal
marker LAMP-1 (red). (C and G) An overlay of hArl8b-GFP and
LAMP-1, in addition to blue Hoechst 33342 staining for visualization of
DNA. (D and H) Detailed view from panels C and F, respectively, as
indicated by the marked areas in panels C and F. Bar, 25 m. (I) hArl8b-
GFP-transfected cells were counted (A to H), and the percentage of cells
displaying a nonpunctuate distribution, as exemplified in panels E to H,
was calculated. At least 500 transfected cells of each type from three
independent experiments were registered. Error bars indicate the stan-
dard deviations. The P value for the two groups (indicated with an aster-
isk) was calculated to be 0.0143 based on a paired t test.
rate the mechanism through which p53 mediates apoptosis
after hNatC mRNA knockdown, we investigated the transcrip-
tional levels of KILLER/DR5, NOXA, and FAS by quantitative
PCR (Fig. 8B). These are markers of transcriptionally medi-
ated p53-dependent apoptosis (as reviewed by Chipuk and
Green [13]). The levels of KILLER/DR5, NOXA, and FAS
were all significantly increased by hMAK3 knockdown, suggest-
ing that sihMAK3 mediates apoptosis through transcriptional
regulation of apoptosis genes. We did not detect any increase
in levels of mitochondria-associated p53 after hMAK3 knock-
down (data not shown). Thus, apoptosis resulting from hMAK3
knockdown does not seem to be mediated through an increase
in p53 association with the mitochondria. When exogenous
hMak3 was expressed in hMAK3 knockdown cells, phenotype
rescue was observed (Fig. 9). This verified that the observed
phenotypes are indeed due to the loss of hMak3.
hArl8b is dependent on hMak3-mediated acetylation for
lysosomal localization. It was recently shown that the human
Arf-like GTPase hArl8b was N-terminally acetylated and
that the acetylated methionine was essential for its lysoso-
mal localization (20). However, the human NAT responsible
for the acetylation was not identified. As the N terminal of
hArl8b is MLALI, we investigated whether hArl8b is an
hMak3 substrate. Our in vitro results show that the hArl8b
N-terminal sequence is indeed a substrate for hMak3-medi-
ated acetylation (Fig. 5).
In HeLa cells subjected to knockdown of hMAK3 expres-
sion, a shift in hArl8b-GFP localization was observed (Fig. 10).
In cells treated with control siRNA, hArl8b-GFP displayed a
punctuate distribution, as previously published (20), overlap-
ping the lysosomal marker LAMP-1 (Fig. 10D). When treated
with sihMAK3, a significant fraction of the observed cells dis-
played aberrant localization of hArl8b-GFP, including loss of
punctuate distribution and the formation of aggregate-like
structures (Fig. 10E to H).
Protein N-terminal acetylation is a very common event in
eukaryotic cells, occurring on a majority of proteins (9). How-
ever, compared to the yeast homologues, the human protein
N-terminal acetyltransferases have not been fully identified or
extensively characterized. In this study, we describe the human
homologues of the yeast NatC complex. Our candidate sub-
units, hMak3, hMak10, and hMak31, stably interact with each
other, localize to the cytoplasm, and associate with ribosomes.
Furthermore, our semipurified hMak3 displays in vitro N-ter-
minal acetyltransferase activity toward peptide substrates
matching the observed substrate specificity of the yeast NatC
complex, displaying a high degree of substrate specificity. In-
terestingly, Munro and colleagues showed in a previous study
that hNat12/hMak3 functionally complemented yMak3p in
mak3 yeast strains (12). Taken together, these findings
strongly argue that hMak3 (hNat12) is the human homologue
of yeast Mak3p. Based on these observations, we suggest that
hMak3, hMak10, and hMak31 constitute the human NatC
complex and that hMak3 is the catalytic subunit of the com-
plex. Interestingly, the hMak3 protein (362 amino acids) is 176
amino acids larger than the yeast Mak3p. This is mainly due to
an N-terminal region not shared by the yeast homologue. Sim-
ilarly, the Arabidopsis thaliana Mak3 also contains additional
residues compared to yeast Mak3, and AtMak3 displays enzy-
matic activity independent of AtMak10 (26). The function of
the N-terminal hMak3 domain is not known. Using the ELM
functional site prediction resource ( (31),
the N-terminal region was found to contain several potential
phosphorylation sites, making it a possible region for post-
translational regulation of hMak3 activity. Similarly, the cata-
lytic subunit of hNatA, hArd1, has a C-terminal domain that is
phosphorylated to different extents depending on cell culture
conditions (24).
All the hNatC subunits localize to the cytoplasm and bind
ribosomes. However, hMak31 also significantly localizes to the
nucleus (Fig. 3). The nuclear localization of hMak31 would be
expected, as its small size allows it to pass through the nuclear
pore complexes. hMak31 (Lsmd1) belongs to the Sm and Sm-like
proteins, which associate with RNA and are often involved in
RNA processing events. Thus, hMak31 may have a nuclear role
linked to RNA processing independent of the hNatC complex.
hMak3, hMak10, and hMak31 are all present, both in a ribosome-
bound and an unbound state. This pattern is also observed for
TABLE 1. Overview of human protein NATs
Yeast NAT
Yeast substrates
(N termini)
NAT subunit
Human homologue(s) and
or source
NatA Ser-, Thr-, Ala-, Gly- Ard1p (catalytic subunit) hArd1, ARD1A, TE2 2, 10, 36
hArd2, ARD1B 4
Nat1p (auxiliary subunit) NATH/hNat1, Tubedown 2, 10, 17, 19
hNat5/hSan 3, 18
NatB Met-Asp-, Met-Glu-, Met-Asn- Nat3p (catalytic subunit) hNat3, hNat5 1, 34
Mdm20p (auxiliary subunit) hMdm20 34
NatC Met-Leu-, Met-Ile-, Met-Phe-, Met-Trp- Mak3p (catalytic subunit) hMak3, hNat12 This study
Mak10p (auxiliary subunit) hMak10 This study
Mak31p (auxiliary subunit) hMak31, Lsmd1 This study
NatD Ser-Gly-Gly-, Ser-Gly-Arg- Nat4p
Listed homologues are based on experimental data. Potential homologues based on database predictions are not included.
Nat5p is predicted to be an acetyltransferase with specific and unknown substrates. Nat5p is not required for NatA activity.
—, no information available.
subunits in the hNatA (T. Arnesen et al., submitted for pubica-
tion) and the hNatB complexes (34). In addition, there seems to
be some difference between hMak3, hMak10, and hMak31 with
respect to the ratio of ribosome-bound versus unbound protein.
In particular, a larger fraction of hMak10 seems to be ribosome
bound compared to hMak3 and hMak31. One could hypothesize
that hMak10 may function as an anchor for ribosomal binding of
the complex, similar to what is observed for hNat1 in the hNatA
In a previous study, Nat13/Nat5 was suggested to be the
vertebrate homologue of yMak3p (38). However, Nat13/Nat5
is the vertebrate protein most similar to yeast Nat5p. Both in
humans and in yeast, Nat5 is described to be a subunit of the
NatA complex (3, 18). To our knowledge, Nat5p has not been
physically linked to the yeast NatC complex or functionally
associated with NatC activity. In our present study we were not
able to demonstrate an interaction between hNat13/hNat5 and
hMak10. Whether hNat13/hNat5 is capable of binding to the
human NatC is therefore doubtful, but more thorough exper-
imentation is needed to settle this issue.
FIG. 11. Schematic representation of functions associated with the human NatC complex. (A) The hNatC complex, composed of the subunits
hMak3, hMak10, and hMak31, associates with ribosomes and acetylates nascent Met-Leu and similar polypeptides. One protein of this type, the
Arf-like GTPase hArl8b, depends on the N-terminal acetylation for its localization to lysosomes. (B) Knockdown of hNatC complex subunits by
siRNA most likely reduces the N-terminal acetylation of some NatC-type substrates. Directly or indirectly, this stabilizes and activates p53, which
in turn induces transcription of genes leading to the induction of apoptosis. Apoptosis decreases the protein level of hMak3, while the mRNA level
is unaffected.
Knockdown of hNatC subunits in HeLa cells leads to cell
death and growth arrest. Knockdown cells displayed reduced
metabolic activity and DNA synthesis, an increase in the sub-
fraction representing nuclear fragmentation, an in
crease in double-stranded DNA breaks, and PARP cleavage
indicating caspase-dependent cell death. All three subunits are
important for normal growth, supporting a model where the
observed phenotypes are caused by lack of hNatC-mediated
N-acetylation. Upon knockdown of hMAK3,hMAK10,or
hMAK31 in the colon carcinoma cell lines HCT116 (p53
no apoptosis was induced, in contrast to HCT116 (p53
cells (Fig. 7). This difference in response between the cell
variants strongly indicates that p53 is essential for induction of
apoptosis in hNatC-depleted cells. Our findings suggest that
this effect is mediated mainly through the transcriptional ac-
tivity of p53, since hMAK3 knockdown increased the protein
levels of p53, increased p53 Ser37 phosphorylation, and in-
duced the transcription of p53 downstream death effector
genes (Fig. 8).
NatC is believed to acetylate relatively few substrates com-
pared to NatA in a manner of high amino acid sequence
specificity at the N-terminal region. This could make hMak3 a
potential target for drug-mediated cancer treatment, as previ-
ously discussed for hNatA (6, 8). An important goal of future
studies will be to further elaborate the pathways through which
hMAK3 knockdown mediates p53-dependent apoptosis.
In combination with the previous studies of the hNatA and
hNatB complexes (1, 6, 34) our present results emphasize the
biological importance of protein N-terminal acetylation in hu-
man cells. It should be noted that hMAK3 knockdown cells in
general demonstrated a stronger phenotype than hMAK10 and
hMAK31 knockdown cells. These effects may indicate that
hMak3 has an additional function independent of hMak10 and
hMak31. Leister and coworkers demonstrated that A. thaliana
Mak3 alone is able to functionally replace the yeast NatC
complex (26). Also, in contrast to AtMak3, knockout of AtMak10
did not result in any obvious defects. This indicates that Mak3
can have functions independent of the NatC complex in or-
ganisms higher than yeast.
The oligopeptides MLALI and MLGTG are identical to the
N termini of the predicted human NatC substrates hArl8b and
mTOR, respectively (38). hArl8b was recently described to be
N-terminally acetylated, and an intact N terminus is essential
for its association with lysosomes (20). mTOR was suggested to
be a direct downstream target for NatC acetylation in ze-
brafish. Our in vitro acetyltransferase assays support our hy-
pothesis that both hArl8b and mTOR are substrates for hNatC
in vivo (Fig. 5). Knockdown of hMAK3 in HeLa cells induces a
change in subcellular distribution of hArl8b-GFP. The ob-
served change was statistically significant, but only a fraction of
the cells (15%) display altered hArl8b-GFP localization as a
consequence of hMAK3 knockdown (Fig. 10). The observed
phenotype changes will to some extent be dependent on the
transfection efficiency of sihMAK3: only a fraction of the
hArl8b-GFP-expressing cells will have efficient knockdown of
hMAK3. Among this subpopulation, one would expect a larger
fraction of cells to display aberrant hArl8b-GFP localization.
Furthermore, large-scale proteomics analyses of NAT knock-
down in human cell lines have demonstrated only partial down-
stream N-terminal acetylation effects in agreement with our
observations (9). Combined, these data strongly support that
hArl8b indeed is an hNatC substrate in vivo. This is to our
knowledge the only human NatC substrate where a clear con-
sequence is observed when the N-terminal acetylation is lost.
Also, it is the only known case with a link between a specific
human NAT and a substrate of which N-terminal acetylation
affects the substrate function. Further characterization of func-
tionally important human NatC substrates will be of great
importance in order to understand the overall impact of N-
terminal acetylation, and in particular, N-terminal acetylation
mediated by the hNatC complex.
With the present study, the human homologues of all three
major yeast NAT complexes, NatA, NatB, and NatC, have
been experimentally determined. In Table 1 we present an
overview of the currently known human protein N-terminal
acetyltransferases. The nomenclature of this enzyme class is
under revision (B. Polevoda, T. Arnesen, and F. Sherman,
unpublished data), and the NatC components will be denoted
as follows: Naa30 (Mak3), Naa35 (Mak10), and Naa38
In summary, we here identify hMak3, hMak10, and hMak31
as the subunits of the human NatC complex. Knockdown of
these subunits induces p53-dependent apoptosis. hMak3-me-
diated acetylation is necessary for the lysosomal localization
and function of hArl8b (Fig. 11).
We thank C. Hoff, J. Torsvik, E. Skjelvik, L. Vikebø, and M. Algrøy
for technical assistance.
This work was supported by The Norwegian Cancer Society, The
Meltzer Foundation, and Norwegian Health Region West.
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    • "Knockdown of NAA30 further influenced the p53 pathway, expression of ribosomal proteins and the expression of glial fibrillary acidic protein (GFAP). Another study showed that the knockdown of NAA30 and the whole NatC complex in HeLa cells caused p53- dependent apoptosis and aberrant localization of ARL8B [7]. It is interesting that the research that was conducted through the seventies and nineties in yeast could be so relevant for understanding the biology of cancer. "
    Full-text · Article · Feb 2016 · Proteomics
    • "There are few known substrates of NAT-C, but based on the Nt-amino acid composition of target proteins NAT-C can potentially Nt-acetylate up to 14.5 % of all cytoplasmic human proteins [47]. The N-terminus of MTOR protein begins with Met-Leu and is a strong candidate as a direct substrate [23, 24]. In a study in zebrafish TOR was suggested as a downstream target of NAT-C [24] . "
    [Show abstract] [Hide abstract] ABSTRACT: 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. 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. 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. 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.
    Full-text · Article · Aug 2015
    • "This observation is in accordance with the fact that in vitro recombinant hNaa60 also displayed very similar substrate specificity profiles as compared to recombinant hNaa50 [7,46], indicative for a potential in vivo substrate redundancy between hNaa50 and hNaa60. Interestingly, the property of Nt-acetylating Met-hydrophobic N-termini is also partially shared with NatC [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Co-translational N-terminal (Nt-)acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2015
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