Characterization of ARD1 variants in mammalian cells

doi:10.1016/j.bbrc.2005.12.018

Biochemical and Biophysical Research Communications

Volume 340, Issue 2, 10 February 2006, Pages 422-427

https://doi.org/10.1016/j.bbrc.2005.12.018 Get rights and content

Abstract

Mouse ARD1 (mARD1) has been reported to negatively regulate the hypoxia-inducible factor 1α (HIF-1α) protein by acetylating a lysine residue and enhancing HIF-1α ubiquitination and degradation. However, it was recently reported that human ARD1 (hARD1) does not affect HIF-1α stability. To further explore the activities of the two orthologs, three mouse (mARD1¹⁹⁸, mARD1²²⁵, mARD1²³⁵) and two human (hARD1¹³¹, hARD1²³⁵) variants were identified and characterized. Among these, mARD1²²⁵ was previously reported as a novel negative regulator of HIF-1α. Amino acid sequence analysis showed that the C-terminal region (aa 158–225) of mARD1²²⁵ completely differs from those of mouse and human ARD1²³⁵, although all three proteins share a well-conserved N-acetyltransferase domain (aa 45–130). The effects of ARD1 variants were evaluated with respect to HIF-1α stability and acetylation activity. Interestingly, mARD1²²⁵ strongly decreased the level of HIF-1α and increased the extent of acetylation, whereas mARD1²³⁵ and hARD1²³⁵ variants had a much weaker effect on HIF-1α stability and acetylation. These results suggest that ARD1 variants might have different effects on HIF-1α stability and acetylation, which may reflect diverse biological functions that remain to be determined.

Section snippets

Materials and methods

Reagents and antibodies. MG132 was purchased from Calbiochem. Mouse anti-HIF-1α antibody was purchased from BD Pharmingen. Anti-GFP and acetyl-lysine antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling, respectively. A polyclonal antibody to ARD1 was produced by Dinona. The immunogens correspond to amino acids 1–17 of mARD1²²⁵ (GenBank Accession No. BC027219) and hARD1²³⁵ (GenBank Accession No. NM_003491). Antibody specificity was confirmed using siRNA targeting ARD1 mRNA

Identification of mammalian ARD1 variants

A search of the NCBI database and DNA sequence alignment allowed identification of mouse ARD1 variants (Fig. 1A). The mARD1¹⁹⁸ (GenBank Accession No. AK078700), mARD1²²⁵ (GenBank Accession No. BC027219), and mARD1²³⁵ (GenBank Accession No. NM_019870) sequences encode proteins of 198, 225, and 235 amino acids, respectively. The mARD1²²⁵ and mARD1²³⁵ proteins have the well-conserved N-acetyltransferase domain (aa 45–130), although mARD1¹⁹⁸ has only a partial domain. Human ARD1 variants were

Discussion

We previously reported that mouse ARD1 interacts with the ODD domain of HIF-1α and mediates acetylation of Lys532, thereby enhancing HIF-1α ubiquitination and degradation [9]. The ARD1 protein that negatively regulates HIF-1α is mARD1²²⁵ (GenBank Accession No. BC027219), which shares the N-terminal region (aa 1–157) with mARD1²³⁵, previously reported as GenBank Accession No. NM_019870. In contrast to our findings, recent studies have shown that hARD1²³⁵ does not affect HIF-1α stability or

References (18)

N. Sugiura et al.
An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development
J. Biol. Chem.
(2003)
A.F. Neuwald et al.
GCN5-related histone N-acetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein
Trends Biochem. Sci.
(1997)
J.W. Jeong et al.
Regulation and destabilization of HIF-1α by ARD1-mediated acetylation
Cell
(2002)
T.S. Fisher et al.
Analysis of ARD1 function in hypoxia response using retroviral RNA interference
J. Biol. Chem.
(2005)
R. Bilton et al.
Arrest-defective-1 protein, an acetyltransferase, does not alter stability of hypoxia-inducible factor (HIF)-1α and is not induced by hypoxia or HIF
J. Biol. Chem.
(2005)
M.K. Bae et al.
Jab1 interacts directly with HIF-1α and regulates its stability
J. Biol. Chem.
(2002)
V. Brenner et al.
Genomic organization of two novel genes on human Xq28: compact head to head arrangement of IDHγ and TRAPδ is conserved in rat and mouse
Genetics
(1997)
F.J.S. Lee et al.
N^α acetylation is required for normal growth and mating of Saccharomyces cerevisiae
Bacteriology
(1989)
J.R. Mullen et al.
Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast
EMBO J.
(1989)

There are more references available in the full text version of this article.

Cited by (37)

The role of N-acetyltransferases in cancers
2024, Gene
Cancer is a major global health problem that disrupts the balance of normal cellular growth and behavior. Mounting evidence has shown that epigenetic modification, specifically N-terminal acetylation, play a crucial role in the regulation of cell growth and function. Acetylation is a co– or post-translational modification to regulate important cellular progresses such as cell proliferation, cell cycle progress, and energy metabolism. Recently, N-acetyltransferases (NATs), enzymes responsible for acetylation, regulate signal transduction pathway in various cancers including hepatocellular carcinoma, breast cancer, lung cancer, colorectal cancer and prostate cancer. In this review, we clarify the regulatory role of NATs in cancer progression, such as cell proliferation, metastasis, cell apoptosis, autophagy, cell cycle arrest and energy metabolism. Furthermore, the mechanism of NATs on cancer remains to be further studied, and few drugs have been developed. This provides us with a new idea that targeting acetylation, especially NAT-mediated acetylation, may be an attractive way for inhibiting cancer progression.
The biological functions of Naa10 - From amino-terminal acetylation to human disease
2015, Gene
Citation Excerpt :
The expression pattern of Naa10 isoforms was analyzed by western blotting in the human cervical adenocarcinoma HeLa, fibrosarcoma HT1080, in the lung adenocarcinoma H1299 cell line, as well as in the murine fibroblast cell line NIH3T3. hNaa10235 was identified as the major form in the human cell lines, whereas mNaa10235 and mNaa10225 were both detected in the murine cell line (Kim et al., 2006), indicating, that – at least in human cells – the shorter Naa10 variant plays only a minor role in hypoxia response. Other studies suggest that the regulation of HIF-1α by Naa10 might require other factors/regulators such as deacetylases and/or may be dependent on different signaling pathways.
N-terminal acetylation (NTA) is one of the most abundant protein modifications known, and the N-terminal acetyltransferase (NAT) machinery is conserved throughout all Eukarya. Over the past 50 years, the function of NTA has begun to be slowly elucidated, and this includes the modulation of protein–protein interaction, protein-stability, protein function, and protein targeting to specific cellular compartments. Many of these functions have been studied in the context of Naa10/NatA; however, we are only starting to really understand the full complexity of this picture. Roughly, about 40% of all human proteins are substrates of Naa10 and the impact of this modification has only been studied for a few of them. Besides acting as a NAT in the NatA complex, recently other functions have been linked to Naa10, including post-translational NTA, lysine acetylation, and NAT/KAT-independent functions. Also, recent publications have linked mutations in Naa10 to various diseases, emphasizing the importance of Naa10 research in humans. The recent design and synthesis of the first bisubstrate inhibitors that potently and selectively inhibit the NatA/Naa10 complex, monomeric Naa10, and hNaa50 further increases the toolset to analyze Naa10 function.
Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features
2012, Molecular and Cellular Proteomics
N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.
Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α
2010, Molecular Cell
To survive in hypoxic environments, organisms must be able to cope with redox imbalance and oxygen deficiency. The SIRT1 deacetylase and the HIF-1α transcription factor act as redox and oxygen sensors, respectively. Here, we found that SIRT1 binds to HIF-1α and deacetylates it at Lys674, which is acetylated by PCAF. By doing so, SIRT1 inactivated HIF-1α by blocking p300 recruitment and consequently repressed HIF-1 target genes. During hypoxia, SIRT1 was downregulated due to decreased NAD⁺ levels, which allowed the acetylation and activation of HIF-1α. Conversely, when the redox change was attenuated by blocking glycolysis, SIRT1 was upregulated, leading to the deacetylation and inactivation of HIF-1α even in hypoxia. In addition, we confirmed the SIRT1-HIF-1α interaction in hypoxic mouse tissues and observed in vivo that SIRT1 has negative effects on tumor growth and angiogenesis. Our results suggest that crosstalk between oxygen- and redox-responsive signal transducers occurs through the SIRT1-HIF-1α interaction.
Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer
2008, Cancer Letters
Citation Excerpt :
The human homolog of yeast Ard1p, ARD1, interacts with NATH to form a functional NAT (N-acetyltransferase) [6], In addition, ARD1 functions as a negative regulator of the hypoxia-inducible factor 1α (HIF-1α). The acetylation of hypoxia-inducible factor 1α (HIF-1α) by a mouse variant of ARD1 (mARD1225) plays an important role in HIF-1α degradation via the ubiquitin-proteasome system [7,8]. However, some researches show that human ARD1 overexpression or silencing has no impact on HIF-1α protein stability [9–11].
Arrest defective 1 (ARD1) is an acetyltransferase involved in cell cycle control in yeast. ARD1 interacts with human N-acetyltransferase (NATH) to form a functional N-terminal acetyltransferase complex. Recently it had been linked with proliferation and apoptosis in mammalian cells, but its function in cancer development remains unclear. To evaluate significance of ARD1 expression in human colorectal cancer, we generated a panel of monoclonal antibodies (mAbs) with high specificity and sensitivity against ARD1. All of the 10 different clones could be used in ELISA and Western blot, and clone 10C12, 13G2, and 4D10 can interact with ARD1 in eukaryotic cells by immunoprecipitation (IP). Clones of 14D4 and 10C12 were strongly reacted to ARD1 in immunocytochemistry (ICH) and immunohistochemistry (IHC). ARD1 expression was evaluated in human colorectal cancer and colitis tissues by immunohistochemical analysis with mAb 14D4. Forty-one were ARD1-positive in 50 colorectal cancer tissues and only 12 were weak positive in the 50 matched normal tissues (P < 0.001). Moreover, ARD1 expression was not detectable in 20 cases of colitis tissue (P < 0.001). Furthermore, all of the six human colorectal cancer cell lines we examined were also ARD1-positive at mRNA and protein levels. Taken together, the novel mAbs against ARD1 we generated could be good tools for both basic and clinical studies, and ARD1 could be a potential biomarker in colorectal cancer.
Hypoxia-inducible factors: Crosstalk between their protein stability and protein degradation
2007, Cancer Letters
Citation Excerpt :
Oddly, human ARD1 (hARD1) potentially interacts with HIF-1α in vivo and in vitro, while it cannot acetylate HIF-1α [34]. One explanation of these interesting phenomena is that mouse ARD1 (mARD1225), which is previously identified as a negative regulator of HIF-1α, has an entirely different C-terminal region (aa 158–225) in contrast with other mouse and human ARD1 variants [35]. Recently, CTGF/CCN2 (connective tissue growth factor) was discovered to influence HIF-1α acetylation indirectly.
As a transcription factor family dependent of oxygen, hypoxia-inducible factors (HIFs) play important roles in organic and cellular homeostasis, which are master regulators of tumorigenesis, angiogenesis and embryogenesis. Up to date, some known posttranslational modifications, such as hydroxylation, acetylation, phosphorylation and S-nitrosylation, have been proved to influence protein stability and transcriptional activity of HIFs. On the other hand, HIFs can be physiologically degraded through ubiquitin-dependent or -independent proteasome pathway. Two biochemical processes involve in many modulators, which are correlated and compose an intricate mechanism to control the biological function of HIFs systemically. Herein, we review and discuss the diverse roles of involved modulators in both HIFs stability and degradation, describe the potential link between the two molecular scenarios.

View all citing articles on Scopus

^☆: This work was supported by Grant No. FG-2-1 of the 21C Frontier Functional Human Genome Project and the Creative Research Initiatives Program, the Ministry of Science and Technology, Korea (to K.-W. Kim) and by the Post-doctoral Fellowship Program of the Korea Science & Engineering Foundation (KOSEF) (to J.-W. Jeong).

^☆☆: Abbreviations: HIF-1, hypoxia-inducible factor-1; NLS, nuclear localization signal; ODD, oxygen-dependent degradation; RT-PCR, reverse transcription-polymerase chain reaction; VEGF, vascular endothelial growth factor.

¹: These authors contributed equally to this work.

View full text

Article preview

Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Identification of mammalian ARD1 variants

Discussion

References (18)

An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development

J. Biol. Chem.

GCN5-related histone N-acetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein

Trends Biochem. Sci.

Regulation and destabilization of HIF-1α by ARD1-mediated acetylation

Cell

Analysis of ARD1 function in hypoxia response using retroviral RNA interference

J. Biol. Chem.

Arrest-defective-1 protein, an acetyltransferase, does not alter stability of hypoxia-inducible factor (HIF)-1α and is not induced by hypoxia or HIF

J. Biol. Chem.

Jab1 interacts directly with HIF-1α and regulates its stability

J. Biol. Chem.

Genomic organization of two novel genes on human Xq28: compact head to head arrangement of IDHγ and TRAPδ is conserved in rat and mouse

Genetics

N^α acetylation is required for normal growth and mating of Saccharomyces cerevisiae

Bacteriology

Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast

EMBO J.

Cited by (37)

The role of N-acetyltransferases in cancers

The biological functions of Naa10 - From amino-terminal acetylation to human disease

Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features

Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α

Generation of novel monoclonal antibodies and their application for detecting ARD1 expression in colorectal cancer

Hypoxia-inducible factors: Crosstalk between their protein stability and protein degradation

Characterization of ARD1 variants in mammalian cells☆,☆☆

Abstract

Section snippets

Materials and methods

Identification of mammalian ARD1 variants

Discussion

J. Biol. Chem.

Trends Biochem. Sci.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Genetics

Nα acetylation is required for normal growth and mating of Saccharomyces cerevisiae

Bacteriology

Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast

EMBO J.

N^α acetylation is required for normal growth and mating of Saccharomyces cerevisiae