|
http://www.abbs.info e-mail:[email protected] ISSN
0582-9879
ACTA BIOCHIMICA et
BIOPHYSICA SINICA 2003, 35(8): 747–751
CN 31-1300/Q |
|
Short Communication |
Interaction
of C17orf25 with ADP-ribose Pyrophosphatase NUDT9 Detected via Yeast Two-hybrid
Method
ZHANG
Hui-Tang, YAN Zhi-Qiang, HU Xiao-Bo, YANG Sheng-Li, GONG Yi*
( Research Center of
Biotechnology, Shanghai Institute for Biological Sciences, the Chinese Academy
of Sciences, Shanghai 200233, China )
Abstract The gene C17orf25 was isolated from the liver by RACE PCR. nudt9
gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA
library. NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose
as its substrate. Fusion expression of C17orf25 and GFP and computer analysis
showed that C17orf25 was probably located in mitochondria. Furthermore,
C17orf25 may suppress the cell growth by interaction with NUDT9.
Key
words C17orf25; NUDT9; yeast
two-hybrid; mitochondria; ADP-ribose
The yeast two-hybrid system has been
developed to provide a powerful genetic approach for identifying protein-protein
interactions in vivo[1－4]. It depends on the
functional assembly of a transcription factor in yeast nucleus as a result of
two proteins’ interacting. This approach is relatively easy in vivo for the
discovery of unknown protein interactions in living cell and several kinds of
yeast hybrid systems have been established[5,6].
C17orf25 (Chromosome 17 open reading
frame 25; Accession No: AF177342) was isolated from the deletion region on
chromosome 17p13.3[7–9]. It encodes a protein of
313 amino acids with a calculated molecular weight 34.7 kD. The Northern blot
results of C17orf25 cDNA and human multiple-tissue showed a single transcript
of ～1.8 kb in human heart, brain,
liver, kidney, pancreas and placenta, but no expression in skeletal muscle and lung
tissues. The transfection of C17orf25 into the hepatocellular carcinoma cell
SMMC7721 and overexpression could inhibit the cell growth. According to the
sequence analysis, C17orf25 has a 42% homologue with the hypothetical
glyoxalase in Caenohabditis elegans, and 27% with the glyoxalase in Brassica
olerecea.
1 Materials
and Methods
1.1 Materials
Gene C17orf25 was obtained from State Key
Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute; E. coli BL21(DE3),
DH5α, HB101 and plasmids pT7450, pT7473(derived from pET21a) are reserved in
the lab; BL21 CodonPlus (DE3)-RIL (genotype: E. coli B F－ ompT hsdS(r－B m－B)
dcm+ Tetr gal λ (DE3) endA Hte [argU ileY
leuW Camr]) was purchased from Novagen; HF7c(genotype: MATa, ura3-52, his3-200,
lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542,
LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::(GAL 17mers) 3-Cyc1TATA-lacZ) and
AH109(genotype: MATa, trp-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ,
LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2;
URA3::MEL1UAS-MEL1TATA-lacZ, MEL1) yeast strains and X-α-gal were purchased
from Clontech; library screening was performed with a human HeLa cDNA library
in pGAD GH from Clontech; yeast nitrogen base (YNB) w/o amino acid was purchased
from Difco; plasmid vectors (i.e., pGBT9 as bait vector; pTD1-1, pVA3-1 pCL1 as
positive controls; pLAM5’-1 as negative control; and pEGFP-C2) were purchased
from Clontech; electroporation was carried out with a “Gene Pulser II”(Bio-Rad, Munich, Germany); NTA-Ni2+
Sepharose was purchased from Bocai Company(Shanghai, China); FolyFect was
purchased from Qiagen.
1.2 Methods
1.2.1 Plasmid
construction C17orf25 gene was constructed
into plasmids pGBT9, pT7450 and pEGFP-C2 between EcoRI and SalI sites. nudt9
gene was cloned into plasmid pT7473 (containing 6 His-tag before the N
terminus) between BamHI (generate the same cohesive end with BglII) and SalI
sites. The primers listed in Table 1 were synthesized by Sangon
(Shanghai, China)
.Table 1 Primer pairs used for cloning and
addition of restriction sites
|
Primer |
Orientation |
Restriction sites |
Sequence |
|
5’ C17orf25 |
forward |
EcoRI |
5’-G CGT GAA TTC GCT
GCT CGC AGA GCT C-3' |
|
3’ C17orf25 |
reverse |
SalI |
5’-G CAT GTC GAC TTA
ACC TGA AGC TTT GGG-3' |
|
5’ Nudt9 |
forward |
BglII |
5'-AGA TCT GTG ACT
ATC AGG TCC TC-3' |
|
3’ Nudt9 |
reverse |
SalI |
5'-GTC GAC TAC AAC
GCA TGG CAG TC-3' |
|
5’GAL4 AD |
forward |
|
5’-GAT GAA GAT
ACC CCA CCA AAC-3’ |
|
3’GAL4 AD |
reverse |
|
5’-ACT TGC GGG
GTT TTT CAG TAT C-3’ |
The
underline parts are restriction sites.
1.2.2 Electroporation[10] A yeast HF7c colony was
inoculated in YPD medium and incubated overnight at 30 ℃ shaking at 300 r/min. Thereafter medium was
added to a final volume of approx 300 mL at an A600 of 0.3. These cells were
further grown to an A600 of 1.2－1.5, and then pelleted by
centrifugation at 10 000 r/min for 10 s followed by two
resuspension/centrifugation cycles in ice-cold water. Thereafter yeast cells
were resuspended in 50 mL 1 mol/L ice-cold sorbitol and centrifuged. Then the
pellet was resuspended in 100 μL 1 mol/L sorbitol to 109
cells/mL (A600≈33). Electroporation was
carried out with a “Gene Pulser II”. Electroporator cuvettes (BioRad) with a
plate distance of 0.2 cm were used throughout the study. Parameters for
electroporation were set at 200 Ω, 25 μF, 1.5 kV, leading to a pulse time of
approximately 4－5 ms.
1.2.3 In vitro
protein interaction test by His-tag pull down method NUDT9 and C17orf25 proteins were
expressed in E. coli and formed inclusion body. Inclusion bodies were dissolved
in 8 mol/L urea and renatured in 1 L Tris-HCl (pH 8.0). C17orf25 and NUDT9 were
mixed together at 4 ℃ for 30 min. 200 μL NTA-Ni2+ Sepharose was filled in a 1 mL
syringer and rinsed with 10 volumes of NTA-0 buffer (20 mmol/L Tris-HCl, pH
7.9, 0.5 mol/L NaCl, 10% glycerol, 0 mmol/L imidazole), then the protein
mixture was loaded to NTA-Ni2+ Sepharose column. Elute the column with 5
volumes of NTA-0, NTA-50 (NTA-0 with 50 mmol/L imidazole), NTA-150(NTA-0 with
150 mmol/L imidazole), NTA-1000 (NTA-0 with 1000 mmol/L imidazole) in turn.
Collect the eluted fraction and perform SDS-PAGE. The negative control only
with C17orf25 protein was done under the same procedures.
1.2.4 Construction
and subcellular localization of fusion protein from pEGFP-C2-C17orf25 C17orf25 gene was constructed into
pEGFP-C2 between EcoRI and SalI sites, which was confirmed by DNA sequencing.
The constructed pEGFP-C2-C17orf25 was transfected into SMMC7721 cells using
Qiagen transfection kit. SMMC7721 cells were seeded at a density of 8×105 cells/disk in a 60 mm dish and then
cultured overnight. Monolayer SMMC7721 cells were grown in RPMI 1640 medium
supplemented with 10% (V/V) heat-inactivated fetal bovine serum (FBS), and
incubated in an incubator with humidified 5% CO2 / 95% air mixture at 37 ℃. Before transfection, the cells were
washed with fresh cell growth medium. The constructed plasmid (2.5 μg) and 10 μL
PolyFect transfection reagent (Qiagen) were mixed for 15 min, and then added to
each well with cells. The cells were co-incubated with the transfection reagent
for 24 h. pEGFP-C2 vector was also transfected into cells in separate wells
under the same condition as a control. The GFP fusion protein was visualized by
laser confocal microscope (Zeiss).
2 Results
2.1 The yeast two-hybrid screen of C17orf25 against cDNA library
First, pGBT9-C17orf25 was transformed
into yeast HF7C. Then, the HeLa cDNA library was transformed into HF7C
containing pGBT9-C17orf25 on SD (trp-, leu-) plate. All colonies were washed
with sterile water and then plated onto another SD (trp-, leu-, his-) plate.
Screen all colonies by β-galactosidase assay and
culture the bluest colony on SD plate. The plasmid was extracted from the yeast
and then transformed into E. coli HB101. After identifying the plasmid by PCR
and restriction enzyme cutting, we sequenced the library plasmid pGAD GH
containing the gene nudt9. We constructed C17orf25 into pGBKT7. Then
pGBKT7-C17orf25 and pGAD GH-nudt9 were co-transformed into AH109 and plated on
SD (trp-, leu-, his-, X-α-gal). After about five days, blue colonies appeared
on the plate as show in Fig.1. The result showed that C17orf25 and NUDT9
can interact in vivo.
Fig.1 H109 colonies containing
plasmids pGBKT7-C17orf25 and pGAD GH-nudt9 on SD (trp－, leu－, his－, X-α-gal) plate grew and appeared blue
2.2 His-tag pull down assay
NUDT9 protein was expressed in E. coli
with a 6 His-tag ahead on it, so that NUDT9 could be attached on the Ni2+
column. C17orf25 and NUDT9 were renatured by dialysing in the 20 mmol/L
Tris-HCl (pH 8.0) buffer. The molecular weight of NUDT9 (without signal
peptide) plus 6 His-tag should be 38.2 kD, but the resulted recombinant NUDT9
protein showed a little bigger on the SDS-PAGE because the 6 His-tag with extra
positive charge could not be covered fully by SDS. We eluted the column with an
imidozole gradient buffer. When eluted with buffer NTA-150 containing 150
mmol/L imidazole, NUDT9 dissociated from the NTA-Ni2+ agarose and bound
C17orf25 (Fig.2, lane 3). When C17orf25 was loaded on the column alone, it
could be eluted with a very low dose of Imidazole at 50 mmol/L (Fig.2,
lane 7). In this assay, it can be concluded that these two proteins do interact
in vitro.
Fig.2 The His-tag pull down assay of
proteins C17orf25 and NUDT9
1－4, samples are C17orf25 and NUDT9; 5,
protein molecular marker; 6－9, sample are C17orf25. The
samples are eluted with the following different buffers: 1, NTA-0 buffer (20
mmol/L Tris-HCl, pH 7.9, 0.5 mol/L NaCl, 10% glycerol, 0 mmol/L imidazole); 2,
NTA-50 buffer (NTA-0 with 50 mmol/L imidazole); 3, NTA-150 buffer (NTA-0 with
150 mmol/L imidazole); 4, NTA-1000 buffer (NTA-0 with 1000 mmol/L imidazole);
6, NTA-0 buffer; 7, NTA-50 buffer; 8, NTA-150 buffer; 9, NTA-1000 buffer.
2.3 Subcellular localization of protein C17orf25
Analysis of the C17orf25 protein sequence
with MitoProt II 1.0a4 (available at,
http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter) revealed that
there was a 0.9593 probability of exporting to mitochondria and the N-terminal
sequence “MAARRALHFVFKVGNRF” would be cleaved[11]. TargetP tool
(available at, http://www.cbs.dtu.dk/services/TargetP/) prediction gives a
score of 0.908 for the mitochondrial targeting peptide in C17orf25 and the mitochondrial
location reliability class is 2 which means the probability is between 0.6 and
0.8[12]. SMMC7721 cells transfected with plasmid pEGFP-C2-C17orf25 were
visualized by laser confocal microscope. The photos showed that GFP tagged
C17orf25 may be concentrated in the mitochondria, while in the control, the GFP
protein distributed homogeneously in the cytoplasm and nulceus (Fig 3).
Fig.3 C17orf25 fusion protein in cell SMMC7721
(A)
GFP tagged C17orf25 in cells in division. (B) GFP tagged C17orf25 in the mature
cells. (C) GFP distributed in the whole cells.
3 Discussion
The yeast two-hybrid screening was
performed using a Clontech HeLa cell cDNA library, NUDT9 protein was obtained
that could interact with C17orf25, and their interaction was confirmed by
His-tag pull down in vitro. Although the exact function of C17orf25 is not
known, its homologue glyoxalase I has been found to catalyze the conversion of the
hemithioacetal of toxic methylglyoxal and glutathione to nontoxic
(S)-D-lactoylglutathione, which might mediate the anti-proliferative
effects[13,14].
NUDT9, a member of the nudix hydrolase
family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase[15].
NUDT9 protein specifically hydrolyses ADP-ribose and IDP-ribose to the
corresponding nucleoside 5′-monophosphates and ribose
5-phosphate[16]. ADP-ribose is a product of NAD hydrolysis and a breakdown
product of the calcium-release second messenger cyclic ADP-ribose[17].
ADP-ribose also acts as protein mono-(ADP-ribose) units in the cells and the
product of poly(ADP-ribose) in higher organisms [16]. Ohlrogge et al.[18] found
that the transfer of the ADP-ribose moiety from NAD onto extracellular arginine
residues of T-cell membrane proteins was mediated by
glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to
ecto-NAD blocked T-cell activation and induced its apoptosis. Ziegler[19]
reviewed the ADP ribosylation as a kind of protein covalent modification. In
the presence of pyrophosphate ADP-ribose liberated by poly ADP-ribose
glycohydrolase might be converted to ATP and ribose phosphate by a
pyrophosphorylase. He conceived that the role of poly (ADP-ribosyl)ation might
be related to both a signaling function and concentration of readily available
cellular energy resources on repair during recovery from DNA damage. NUDT9
pyrophosphatase hydrolyses ADP-ribose to AMP and ribose 5-phosphate. It can
regulate the concentration of ADP-ribose and may also help produce energy
resources.
Mitochondria are the sites of respiration
in eukaryote cells. They are involved in the cell apoptosis as discovered in
recent ten years. Now mitochondria are considered to be central coordinators of
cell death and early apoptotic process particularly[20]. Lin et al.[16] and
Perraud et al.[15] revealed that NUDT9 located in mitochondria. In our paper it
was shown that protein C17orf25 may be targeted to mitochondria mitochondria,
which confirmed the interaction of C17orf25 and NUDT9. So the interaction may
take place in mitochondria and the effect is related to mitochondria. C17orf25
showed a suppressive effect on SMMC7721 cell growth[7], which might work by
means of NUDT9. The detailed mechanism of C17orf25 in suppressing the cell
growth and the interaction C17orf25 and NUDT9 remains to be elucidated in
further investigation.
Acknowledgements We
would like to thank Prof. Sha Jia-Hao and Dr. Yin Lan-Lan in Nanjing Medical University
for their kind help with the use of laser confocal microscope.
References
1 Chien CT, Bartel PL,
Sternglanz R, Fields S. The two-hybrid system: A method to identify and clone
genes for proteins that interact with a protein of interest. Proc Natl Acad Sci
USA, 1991, 88(21): 9578－9582
2 Estojak J, Brent R,
Golemis EA. Correlation of two-hybrid affinity data with in vitro measurements.
Mol Cell Biol, 1995, 15(10): 5820－5829
3 Fields S, Song O. A
novel genetic system to detect protein-protein interactions. Nature, 1989,
340(6230): 245－246
4 Guarente L. Strategies
for the identification of interacting proteins. Proc Natl Acad Sci USA, 1993,
90(5): 1639－1641
5 Vidal M, Brachmann RK,
Fattaey A, Harlow E, Boeke JD. Reverse two-hybrid and one-hybrid systems to
detect dissociation of protein-protein and DNA-protein interactions. Proc Natl
Acad Sci USA, 1996, 93(19): 10315－10320
6 Putz U, Skehel P, Kuhl
D. A tri-hybrid system for the analysis and detection of RNA-protein interactions.
Nucleic Acids Res, 1996, 24(23): 4838－4840
7 Qin WX, Wan F, Sun FY,
Zhang PP, Han LW, Huang Y, Jiang HQ et al. Cloning and characterization of a
novel gene (C17orf25) from the deletion region on chromosome 17p13.3 in
hepatocelular carcinoma. Cell Res, 2001, 11(3): 209－216
8 Guo JY, Xu J, Mao DQ,
Fu LL, Gu JR, Zhu JD. The promoter analysis of the human C17orf25 gene, a novel
chromosome 17p13.3 gene. Cell Res, 2002, 12(5-6): 339－352
9 Huang Y, Qin WX, Wan
DF, Zhao XT, Gu JR. Computational analysis and prediction for exons of PAC579
genomic sequence. Science in China (Series C), 2001, 44(5): 533－540
10 Helmuth M, Altrock W, Bockers
TM, Gundelfinger ED, Kreutz MR. An electrotransfection protocol for yeast
two-hybrid library screening. Anal Biochem, 2001, 293(1): 149－152
11 Claros MG., Vincens P.
Computational method to predict mitochondrially imported proteins and their
targeting sequences. Eur J Biochem, 1996, 241(3): 779－786
12 Emanuelsson O, Nielsen H,
Brunak S, von Heijne G. Predicting subcellular localization of proteins based
on their N-terminal amino acid sequence. J Mol Biol, 2000, 300(4): 1005－1016
13 Himo F, Siegbahn PE.
Catalytic mechanism of glyoxalase I: A theoretical study. J Am Chem Soc, 2001,
123(42): 10280－10289
14 Ayoub FM, Allen RE,
Thornalley PJ. Inhibition of proliferation of human leukaemia 60 cells by
methylglyoxal in vitro. Leuk Res, 1993, 17(5): 397－401
15 Perraud AL, Shen B, Dunn CA,
Rippe K, Smith MK, Bessman MJ, Stoddard BL et al. NUDT9, a member of the Nudix
hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose
pyrophosphatase. J Biol Chem, 2003, 278(3): 1794－1801
16 Lin S, Gasmi L, Xie Y, Ying
K, Gu S, Wang Z, Jin H et al. Cloning, expression and characterisation of a
human Nudix hydrolase specific for adenosine 5′-diphosphoribose
(ADP-ribose). Biochim Biophys Acta, 2002, 1594(1): 127－135
17 Perraud AL, Fleig A, Dunn CA,
Bagley LA, Launay P, Schmitz C, Stokes AJ et al. ADP-ribose gating of the calcium-permeable LTRPC2 channel
revealed by Nudix motif homology. Nature, 2001, 411(6837): 595－599
18 Ohlrogge W, Haag F, Lohler J,
Seman M, Littman DR, Killeen N, Koch-Nolte F. Generation and characterization
of ecto-ADP-ribosyltransferase ART2.1/ART2.2-deficient mice. Mol Cell Biol,
2002, 22(21): 7535－7542
19 Ziegler M. New functions of a
long-known molecule. Emerging roles of NAD in cellular signaling. Eur J
Biochem, 2000, 267(6): 1550－1564
20 Ravagnan L, Roumier T,
Kroemer G. Mitochondria, the killer organelles and their weapons. J Cell
Physiol, 2002, 192(2): 131－137
__________________________________________
Received: March 24, 2003 cepted: May 23, 2003
This work was supported by a grant from the National Key Technologies R&D Program (No. 2002BA711A021)
*Corresponding author: Tel: 86-21-64700892-369; Fax: 86-21-64700244; e-mail: [email protected]