correction notice · 2012-10-17 · Lian-ru Zhang1,Hong-kui Zhang2, Jian-ping He1, Wei-jia Wang1,...
Transcript of correction notice · 2012-10-17 · Lian-ru Zhang1,Hong-kui Zhang2, Jian-ping He1, Wei-jia Wang1,...
nature chemical biology
corrections Nature chemical biology
the orphan nuclear receptor nur77 regulates LKB1 localization and activates AMPKYan-yan Zhan, Yan Chen, Qian Zhang, Jia-jia Zhuang, Min Tian, Hang-zi Chen, Lian-ru Zhang, Hong-kui Zhang, Jian-ping He, Wei-jia Wang, Rong Wu, Yuan Wang, Chunfang Shi, Kai Yang, An-zhong Li, Yong-zhen Xin, Terytty Yang Li, James Y Yang, Zhong-hui Zheng, Chun-dong Yu, Sheng-Cai Lin, Chawn-shang Chang, Pei-qiang Huang, Tianwei Lin & Qiao Wu
Nat. Chem. Biol.; doi:10.1038/nchembio1069; corrected online 28 September 2012In the version of this article initially published online, the name of the contributing author Chawnshang Chang was misspelled. The error has been corrected for the PDF and HTML versions of this article.
correction notice
Nature Chemical Biology: doi:10.1038/nchembio.1069
Supplementary Information
The orphan nuclear receptor Nur77 regulates LKB1 localization and
activates AMPK
Yan-yan Zhan1,4, Yan Chen1,4, Qian Zhang1,4, Jia-jia Zhuang2, Min Tian1, Hang-zi Chen1,
Lian-ru Zhang1,Hong-kui Zhang2, Jian-ping He1, Wei-jia Wang1, Rong Wu1, Yuan Wang1,
Chunfang Shi1, Kai Yang1,An-zhong Li1, Yong-zhen Xin1, Terytty Yang Li1, James Y Yang1,
Zhong-hui Zheng1, Chun-dong Yu1, Sheng-Cai Lin1, Chawnshang Chang3, Pei-qiang Huang2,
Tianwei Lin1* & Qiao Wu1*
1 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen
University, Xiamen, Fujian 361005, China 2 Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen 361005, Fujian Province, P. R. China 3 George H. Whipple Lab for Cancer Research, Departments of Pathology and Urology, and
the Cancer Center, University of Rochester Medical Center, Rochester, NY 14642, USA
Nature Chemical Biology: doi:10.1038/nchembio.1069
Supplementary Methods
Crystallization, data collection and processing
Crystals of LBD and its complex with TMPA were obtained at 16°C by the method of
hanging drop vapour diffusion. The droplets were consisted of a 1:1 (v/v) mixture of LBD at 7
mg/ml and the well solution of 100 mM sodium citrate, pH 4.2, and 7% PEG6000. LBD and
TMPA were mixed at a molar ratio of 1:10 and the co-crystallization was carried out under the
similar conditions for the apo protein except the addition of 5% DMSO in the well solution.
In both cases, the crystals appeared after 36-48 hours and reached the maximal size after 5
days. The crystals were transfered to cryo-solutions comprised of either the reservoir solution
supplemented with 25% (V/V) glycerol or a solution containing 100 mM MES, pH 6.2, 10%
PEG6000, 25% (v/v) glycerol, and 1 mM TMPA. The crystals were flash-frozen in liquid
nitrogen at -170°C and the diffraction data was collected at 100 K from single crystals using
either the synchrotron radiation at Beamline BL17U1 at Shanghai Synchrotron Radiation
Facility or an in-house X-ray generator (Cu K�, �=1.5418 Å) coupled with a Marresearch
mar345dtb image plate detector, and analyzed using the program AUTOMAR
(http://www.marresearch.com/automar, AUTOMAR User's Guide, v.1.4 (2003)). The apo
crystal diffracted to a resolution of 2.06 Å and the holo crystal diffracted to 2.2 Å. Both apo
and holo crystals belong to the same space group P212121, and have similar unit cell
parameters: a = 74.43 Å, b = 76.77 Å, c = 128.95 Å and a = 74.24 Å, b = 76.77 Å, c = 129.19
Å, respectively.
Data were analyzed by molecular replacement using Phaser1. The search model for
molecular replacement is the crystal structure of apo LBD (PDB code 2QW4; chain A). Two
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LBD molecules were in the asymmetric unit. The programs Refmac5 and Coot in CCP4 suit2-5
were used for refinement and model building. The data statistics are shown in Supplementary
Tab. 2. The coordinates have been deposited to Protein Data Bank. The accession number are
3V3E for the apo structure and 3V3Q for the TMPA bounded structure.
Molecular modeling for LKB1 binding to Nur77
The LKB1 crystal structure was from the Protein Data Bank (accession number 2WTK).
The simulation of LKB1 docking to LBD of Nur77 was performed by Hex docking server
(http://www.loria.fr/~ritchied/hex_server/).
Plasmid constructions
Full-length human EST clones for LKB1 and AMPK�2 were purchased from Addgene
(USA). Human AMPK�1, AMPK�1, and AMPK�2(1-312) (kindly provided by Peng Li,
Tsinghua University, China) were subcloned into different vectors as required, which included
pGEX-4T-1, pET-28a or pCMV5 vectors containing HA-, Flag- or Myc-tags. Constructs of
Nur77 or LKB1 mutants were created by PCR-based mutagenesis and verified by DNA
sequencing. Primer information for the various constructs is available upon request. A
tricistronic expression vector harboring cDNA for all three subunits of AMPK (rat
AMPK�����/����was kindly provided by Jiawei Wu (Tsinghua University, Beijing, China).
The point mutations of Nur77, LBD or LKB1 were generated with the Quick Change
mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions
and verified by sequencing.
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His-LBD used in crystallization and fluorescence quenching covers residues 351-598 of
Nur77. The His tag is at the C-terminus after 2 extra residues from the vector (LEHHHHHH).
The coding sequence of LBD was amplified and subcloned into pET-22b vector (Novagen) by
using the NdeI and XhoI restriction sites.
Antibodies and drugs
Mouse anti-HA (cat. #H-9658), anti-FLAG (cat. #F-3165), anti-�-tubulin (cat. #T4026)
and rabbit anti-PARP (cat. #P-7605) antibodies were purchased from Sigma. Rabbit anti-Myc
(cat. #SC-789), mouse anti-GFP (cat. #SC-9996) antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-GST (cat. #A00865) antibody was
purchased from Genscript Inc. (Piscataway, NJ). Rabbit anti-AMPK� (cat. #2532),
anti-phospho-AMPK�(T172)� (cat. #2535), anti-LKB1 (cat. #3047, #3050),
anti-phospho-LKB1(S428) (cat. #3482), anti-phospho-LKB1(T189) (cat. #3054),
anti-phospho-LKB1(S334) (cat. #3055), anti-phospho-MARK (cat. #4836), anti-Nur77 (cat.
#3562) antibodies were purchased from Cell Signaling Technology. Rabbit
anti-phospho-LKB1(S307) (cat. #09-478) antibody was purchased from Millipore (Billerica,
MA). Rabbit anti-MARK (cat. #PA002279-B1093) antibody was purchased from SYD labs.
Metformin, leptomycin (LMB) and streptozotocin (STZ) were purchased from Sigma.
Knock-down of Nur77 by lentivirus-based RNA interference
The Nur77 knock-down LO2 cell line was generated by using a lentivirus transfection
system6. The human Nur77 shRNA target is 5�-GGGCATGGTGAAGGAAGTT-3�. The
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shRNA control (scramble) sequence is 5�-GGCTACGTCCAGGAGCGCACC-3�.
Oligonucleotides (Invitrogen) were annealed and subcloned into lentiviral vector pLL3.7.
Lentiviruses were generated by cotransfecting subconfluent HEK293T cells with the lentiviral
vector and packaging plasmids by calcium phosphate transfection. Viral supernatants were
collected at 72 h after the transfection, centrifuged at 3,000 g for 15 min, and filtered through
0.45-�m filters (Millipore). Freshly plated LO2 cells were then infected with the lentivirus,
and the Nur77 knockdown efficiency was determined by western blot.
Preparation of nuclear and cytoplasmic fractions from tissue
Fresh liver samples were homogenized in Buffer A (10 mM HEPES, pH 7.9, 10 mM
KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 0.15% NP-40) containing 1% protease inhibitor
and placed in ice for 15 min. The homogenates were centrifuged at 12,000 g for 1 min at 4°C,
and the supernatant (the cytosolic fraction) was stored at -80°C. The pellet was washed three
times with Buffer A and then resuspended in Buffer B (20 mM HEPES, pH 7.9, 0.4 mM NaCl,
1 mM EDTA, 1 mM EGTA, 0.5% NP-40) containing 1% protease inhibitor and sonicated at
4°C. Cellular debris was removed by centrifugation at 12,000 g for 30 min at 4°C, and the
supernatant (the nuclear fraction) was stored at –80°C for further analysis.
Measurement of protein-compound binding affinity by fluorescence quenching assay
Fluorescence spectra were obtained as previously described7, 8. GST- or His-fusion
proteins were expressed in E. coli strain BL21, purified and dialyzed against phosphate buffer
(pH6.2). All three subunits of AMPK �/�/� were produced from a tricistronic transcript for the
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spontaneous formation of the heterotrimeric complex in the bacterial cytosol. For
protein-TMPA interactions, proteins were incubated with various concentrations of TMPA,
and the fluorescence quenching was monitored at 25 °C with a slit width of 5 nm for
excitation, and a slit width of either 5 nm or 2.5 nm for emission. The excitation wavelength
was 280 nm, and the emission spectra were recorded from 285 to 430 nm. To estimate the
binding affinity, the fluorescence intensities at 334 nm with increasing concentrations of
quencher were measured, and the values of Kd were calculated according to the standard
formula3.
Measurement of protein-protein binding affinity using biolayer interferometry
Binding assays were carried out at 25 using the OctetRedTM biolayer interferometry
instrument (ForteBio, Inc.). 10 g/ml of biotinylated His-LBD or its mutants was captured on
the surface of streptavidin optical sensor tips (SA biosensors, ForteBio, Inc.), washed, and
transferred into PBS buffer containing different concentrations of GST-LKB1 proteins (1.778,
0.444, 0.222, 0.111, and 0.0556 M). The reactions were monitored for 10 min and the bound
material was subsequently transferred to PBS buffer. The dissociation reactions were
monitored for 15 min and the data were processed based on the 1:1 model and the average Kd
was extracted from binding results for five different concentrations of the analytes using the
Octet User Software version 3.1.
Circular dichroism spectroscopy
Circular dichroism (CD) measurements were carried out using a Jasco J-810
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spectropolarimeter (JASCO, Tokyo). Proteins were dialyzed against phosphate buffer (10 mM
phosphate, pH 6.2). The CD spectra were obtained using a cell with a 0.5-cm path length.
Coimmunoprecipitation and western blot analysis
Coimmunoprecipitation was performed as described previously9. Briefly, cell lysates
were incubated with the appropriate antibody for 1 hour and subsequently incubated with
protein A-Sepharose beads for another 1 hour. The protein-antibody complexes that were
recovered on beads were subjected to western blot analysis after separation by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The immunoreactive
products were detected using enhanced chemiluminescence (Pierce, Socochim SA, Lausanne,
Switzerland).
Luciferase reporter assay
Cells were transfected with pGL6-NBRE luciferase reporter gene and �-galactosidase
(�-gal) expression vectors. Twenty-four hour after transfection, cells were treated with 0.5 M
TMPA for indicated times. The luciferase activity was measured and normalized for
transfection efficiency to internal �-gal activity.
Immunofluorescent staining
Cells were transfected with different expression plasmids as required. After transfection,
cells were treated with 10 M TMPA for 6 hours. The cells were fixed in 4%
paraformaldehyde, and then incubated with anti-Flag antibody followed by FITC conjugate
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secondary antibody for identification of LKB1 protein. Nuclei were simultaneously stained
with Hoechst. Stained cells were visualized under a confocal microscope (Leica, TCS SPISE).
In vitro GST pull-down assay
GST or GST-fusion proteins were expressed in E. coli strain BL21 and purified using
glutathione-Sepharose (Sigma). The bead-bound GST or GST-fusion proteins were then
incubated with 500 ng His-TR3 protein in 400 l modified ELB buffer (400 mM NaCl, 50
mM Tris-HCl (pH 7.6), 0.5% Nonidet P-40 (NP-40), 1 mM PMSF) at 4°C for 3 h. Unbound
GST or GST-tagged proteins were removed by washing with modified ELB buffer. The
proteins were eluted by boiling in loading buffer for 10 min, resolved by SDS–PAGE, and
examined by western blot analysis with anti-Nur77 antibody.
In vitro kinase assay
The in vitro kinase assay was performed as described elsewhere10. Briefly, Myc-LKB1
proteins that coimmunoprecipitated with anti-Myc antibody from transfected 293T cells and
eluted with Myc peptide were used as kinases. Purified fusion proteins GST-AMPK�(1–312)
or His-TR3 was incubated in a 50 l reaction mixture which contained 25 mM Tris-HCl (pH
7.4), 10 mM magnesium chloride, 5 mM �-glycerol phosphate, 1 mM dithiothreitol (DTT),
0.1 mM sodium orthovanadate, 50 M cold ATP, 10 Ci [�-32P]ATP, and appropriate amount
of myc-LKB1 (about 60 pmol/min protein kinase activity). The kinase mixtures (substrate and
kinase in reaction mixtures) were incubated at 30C for 30 min, terminated with SDS sample
buffer, and then subjected to SDS-PAGE and autoradiography.
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Real-time PCR
Total RNA was extracted using the RNeasy kit (QIAGEN) and reverse transcribed with
M-MuLV reverse-transcriptase (Fermentas). cDNA was amplified using primers specific for
G6pc and Pepck. Real-time PCR was performed using Sybr Green-based detection in
Rotor-Gene according to the manufacturer’s instruction. �-actin levels were used as
normalization controls. The primers were as bellow:
G6pc, 5'-CACCGACTACTACAGCAACAGC-3'
5'-ATCCCAACCACAAGATGACG-3'
Pepck, 5'-CATTGAGGGTATCATCTTTGGTGG-3'
5'-CAGGTATTTGCCGAAGTTGTAGC-3'
�-actin, 5'-CACCAACTGGGACGACATG-3'
5'-GCACAGCCTGGATAGCAAC-3'
Statistical analysis
Data were expressed as the means ± SEM. Differences between two groups were
assessed by a two-tailed unpaired student’s t-test or by a one-way or two-way ANOVA with
SPSS software (version 13; SPSS Inc, chicago, IL). The null hypothesis was rejected at the
level of p < 0.05.
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Synthesis of TMPA
A. Synthetic scheme
MeO
MeO
MeO
Meldrum's acid
TEAF
MeO
MeO
OMe
PPA MeO
MeO
OMe
MeO
MeO
OMeOsO4, NMOCH3SO2NH2
Actone/H2O
NaIO4
THF/H2O2. HCl
NaClO2, NaH2PO4
THF/t-BuOH/H2O
2-Methyl-2-butene
2 3
7
C2H5OH
SOCl2
CHO
O
R6
8 (TMPA)
4
1. RMgBr
CO2H2
R = (CH2)6Me5
MeO
MeO
OMe
R
OH
OH
MeO
MeO
OMe
CO2HR
O
MeO
MeO
OMe
CHOR
O
MeO
MeO
OMe
CO2C2H5R
O1
Supplementary Scheme 1
B. Experimental procedures
General. Melting points were determined on an X-4 digital micro melting point
apparatus without additional correction. Infrared spectra were obtained with a Nicolet Avatar
330 FT-IR spectrometer using film KBr pellet technique. 1H NMR spectra were recorded in
CDCl3 on a Bruker AV400 with tetramethylsilane as an internal standard. Chemical shifts are
expressed in � (ppm) units downfield from TMS. Mass spectra were recorded by a Bruker
Dalton Esquire 3000 plus LC-MS apparatus. Optical rotations were measured with a
Perkin-Elmer 341 automatic polarimeter. THF was distilled over sodium and dichloromethane
was distilled over P2O5 prior to use. Flash column chromatography was carried out with silica
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gel (300-400 mesh) with eluent ethyl acetate/petroleum ether (60-90 C) (EtOAc/PE).
3-(2,3,4-Trimethoxyphenyl)propanoic acid (3). 2, 3, 4-trimethoxybenzaldehyde 2 (1.00
g, 5.09 mmol) was added to a solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s
acid) (0.88 g, 6.12 mmol) in TEAF (9 mL). The mixture was stirred at 95~100 C for 3 h.
After the reaction mixture was cooled to 0 C, ice water (6 mL) was added. The solution was
acidified to pH=1 with 6 N HCl. The mixture was extracted with ethyl acetate (5 × 1 0 mL).
The combined organic phases were washed with brine (0.5 mL) and dried over anhydrous
Na2SO4. After concentrated in vacuo, it was purified by flash column chromatography on
silica gel (EtOAc/PE = 1/2) to give Compound 3 (1.04 g, yield: 85%) as a white solid. Mp
75-77 C (EtOAc/PE); IR (film) �max: 3434, 2971, 2936, 2830, 1709, 1495, 1468, 1416, 1277,
1098, 1051 cm-1; 1H NMR (400 MHz, CDCl3) � 2.64 (t, J = 7.8 Hz, 2H, ArCH2), 2.89 (t, J =
7.8 Hz, 2H, CH2COOH), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.60 (d,
J = 8.5 Hz, 1H, ArH), 6.85 (d, J = 8.5 Hz, 1H, ArH); 13C-NMR (100 MHz, CDCl3) � 179.4,
152.5, 151.9, 142.2, 126.0, 123.7, 107.1, 60.8, 60.7, 55.9, 34.8, 25.1; MS (ESI) m/z 263
(M+Na+, 100%). Anal. Calcd. for C12H16O5: C, 59.99; H, 6.71. Found: C, 59.61; H, 6.87.
4,5,6-Trimethoxy-2,3-dihydroinden-1-one (4). Compound 3 (0.10 g, 0.42 mmol) and
polyphosphoric acid (PPA) (2.00 g) were mixed and stirred vigorously at 50 C for 4 h. Ice
water was added to the mixture by drops (6 mL) at 0 C and the reaction mixture was
extracted with ethyl acetate (5 × 10 mL). The combined organic phases was washed with
brine (0.5 mL) and dried over anhydrous Na2SO4. After concentrated in vacuo, it was purified
by flash column chromatography on silica gel (EtOAc/PE = 1/3) to give Compound 4 (74 mg,
yield: 80%) as a white solid. Mp 82-86 C (EtOAc/PE); IR (film) �max: 3436, 2928, 2843,
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1709, 1601, 1473, 1421, 1347, 1317, 1279, 1236, 1206, 1132, 1094, 1030 cm-1; 1H NMR (400
MHz, CDCl3) � 2.68 (t, J = 5.6 Hz, 2H, ArCH2), 3.05 (t, J = 5.6 Hz, 2H, CH2CO), 3.89 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 7.03 (s, 1H, ArH); 13C NMR (100 MHz,
CDCl3) � 206.0, 154.2, 150.0, 147.6, 141.6, 132.5, 100.6, 61.1, 60.6, 56.2, 36.1, 22.4; MS
(ESI) m/z 245 (M+Na+, 100%).
3-Heptyl-5,6,7-trimethoxy-1H-indene (5). To a solution of Compound 4 (68 mg, 0.65
mmol) in dry CH2Cl2 (40 mL), heptylmagnesium bromide (25 mL, 1.50 M in Et2O) in diethyl
ether was added drop by drop under N2 at 0 C. The mixture was allowed to warm to the room
temperature and stirred for 1.5 h. 6 N HCl (15 mL) was added drop by drop to the mixture at
0 °C. After stirred for 1h, the mixture was extracted with CH2Cl2 (5 × 15 mL). The combined
organic phases were washed with brine (0.5 mL) and dried over anhydrous Na2SO4. After
concentrated in vacuo, the purification was done by flash column chromatography on silica
gel (EtOAc/PE = 1/15) to give the compound 5 (1.87 g, yield: 82%) as a pale yellow oil. IR
(film) �max: 3438, 2928, 2854, 1574, 1464, 1415, 1115, 1040 cm-1; 1H NMR (400 MHz,
CDCl3) � 0.92 (t, J = 5.3 Hz, 3H, CH3), 1.25-1.50 (m, 8H, (CH2)4), 1.65-1.75 (m, 2H,
CH2C5H11), 2.51(t, J = 6.8 Hz, 2H, CH2C6H13), 3.35 (s, 2H, ArCH2) 3.90 (s, 3H, OCH3), 3.94
(s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.18 (s, 1H, CH=C), 6.72 (s, 1H, Ar-H); 13C NMR (100
MHz, CDCl3) � 153.0, 149.3, 144.2, 141.8, 139.2, 127.3, 127.2, 98.6, 61.0, 60.1, 56.3, 35.3,
31.8, 29.5, 29.1, 27.9, 27.6, 22.6, 14.0; MS (ESI) m/z 327 (M+Na+, 100%); Anal. Calcd. for
C19H28O3: C, 74.96; H, 9.27. Found: C, 74.92; H, 9.08.
1-Heptyl-4,5,6-trimethoxy-2,3-dihydro-1H-indene-1,2-diol (6). Compound 5 (0.304 g,
1 mmol) was dissolved in 10 mL of acetone and 1 mL of water. Methane sulfonamide (0.095
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g, 1 mmol) and 4-Methylmorpholine-4-oxide (NMO) (0.140 g, 1.2 mmol) were then added.
The resulting mixture was stirred at 0 °C for 30 min. Osmium tetroxide solution in water (1
mL = 0.004 mmol) was added slowly, and the reaction turned to brownish-yellow. The
reaction was allowed to warm to room temperature and stirred for 24 h. After disappearance
of the reactant (monitored by TLC), the reaction was quenched with 1 g of sodium bisulfate
followed by the evaporation of acetone. The residue was extracted with EtOAc/water (2 × 10
mL/ 10 mL). The organic layers were combined, dried over sodium sulfate, filtered,
evaporated, and purified by was chromatographed over silica with EtOAc/PE (1/3). The
organic fractions were evaporated to provide Compound 6 (0.304 g, yield: 90%) as a colorless
oil. IR (film) �max: 3426, 2931, 1596, 1468, 1116 cm–1; 1H NMR (400 MHz, CDCl3) � 0.84 (t,
J = 6.9 Hz, 3H, CH3), 1.23-1.36 (m, 10H, (CH2)5), 1.57-1.73 (m, 2H, CH2CH3), 2.75 (dd, J =
3.0, 16.3 Hz, 1H, ArCHH), 3.03 (dd, J = 5.8, 16.3 Hz, 1H, ArCHH), 3.30 (s, 1H,
ArCOH-D2O), 3.36 (d, J = 5.9, 1H, ArCH2CHOH-D2O), 3.79 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 4.11 (m, 1H, CHOH), 6.59 (s, 1H, ArH); 13C NMR (100 MHz,
CDCl3) � 13.9, 22.4, 23.6, 29.0, 29.9, 31.6, 35.3, 38.2, 56.0, 60.2, 60.7, 76.3, 82.6, 102.7,
123.4, 140.7, 141.3, 149.6, 153.2; MS (ESI) m/z 361 (M+ Na+, 100%); Anal. Calcd for
C19H30O5: C, 67.43; H, 8.93. Found: C, 67.25; H, 9.35.
2-(2,3,4-Trimethoxy-6-octanoylphenyl)acetaldehyde (7). To a solution of Compound 6
(500mg, 1.5 mmol) in THF/H2O (5 mL/2.5 mL), NaIO4 (316mg, 1.5 mmol) was added and
stirred at room temperature for 4 h. The mixture was added with silica gel (1 g, 100-200 mesh)
and EtOAc (5 mL). After filtration and concentration, it was purified by flash column
chromatography on silica gel (EtOAc/PE = 1/6) to give Compound 7 (430 mg, yield: 85%) as
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a colorless oil. IR (film) �max: 2930, 2855, 1725, 1592, 1138, 1052 cm–1; 1H NMR (400 MHz,
CDCl3) � 0.89 (t, J = 6.9 Hz, 3H, CH3), 1.25-1.32 (m, 8H, (CH2)4), 1.63-1.70 (m, 2H,
CH2CH3), 2.87 (t, J = 7.2, 2H, ArCOCH2), 3.81 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 3.95-3.96 (m, 2H, ArCH2), 7.10 (s, 1H, ArH), 9.75 (s, 1H, CHO); 13C NMR (100
MHz, CDCl3) � 14.0, 22.6, 24.4, 29.1, 29.2, 31.7, 40.8, 41.2, 56.3, 60.8, 61.1, 108.9, 120.8,
133.1, 145.5, 152.0, 153.0, 199.4, 202.5; MS (ESI) m/z 359 (M+ Na+, 100%); Anal. Calcd
for C19H28O5: C, 67.83; H, 8.39. Found: C, 68.13; H, 8.69.
2-(2,3,4-Trimethoxy-6-octanoylphenyl)acetic acid (8). To a stirring solution of
Compound 7 (230 mg, 0.68 mmol) in THF/t-BuOH/H2O (3.2 / 1.2 / 0.4 mL),
2-methyl-2-buene (0.72 mL, 5.5 mmol) and NaH2PO4 (320 mg, 2.05 mmol) was added before
the addition of NaClO2 (185 mg, 2.05 mmol) at 0 °C. The mixture was stirred at room
temperature for 2 h. Saturated NaHSO3 solution (3 mL) was added and the mixture was
extracted with CH2Cl2 (5 × 10 mL). The combined organic phases were washed with brine (5
mL) and dried over anhydrous Na2SO4. After concentrated in vacuo, the purification was
carried out by flash column chromatography on silica gel (EtOAc/PE = 1/3) to give
Compound 8 (223 mg, yield: 93%) as a pale yellow soild. Mp 105-109 oC (EtOAc/PE); IR
(film) �max: 3434, 2959, 2930, 2850, 1703, 1677, 1597, 1405, 1334, 1246, 1226, 1188, 1142,
1106, 1053 cm-1; 1H-NMR (400 MHz, CDCl3) � 0.82 (t, J = 6.9 Hz 3H, CH3), 1.06-1.40 (m,
8H, (CH2)4), 1.53-1.75 (m, 2H, CH2C5H11), 2.89 (t, J = 6.6Hz, 2H, ArCOCH2), 3.77-3.96 (m,
11H, 3OCH3, CH2COOH), 7.05 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) � 203.6, 177.0,
152.9, 152.0, 145.4, 133.2, 121.4, 108.5, 61.2, 60.8, 56.2, 40.9, 32.3, 31.7, 29.2, 29.1, 24.4,
22.6, 14.1; MS (ESI) m/z 375 (M+Na+, 100%). Anal. Calcd. for C19H28O6: C, 64.75; H, 8.01.
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Found: C, 64.84; H, 7.78.
Ethyl 2-(2,3,4-trimethoxy-6-octanoylphenyl)acetate (1 or TMPA). To a solution of
Compound 8 (104 mg, 0.30 mmol) in dry ethanol (1.5 mL), SOCl2 (0.06 mL) was added drop
by drop at 0 C under N2 and the mixture was stirred at room temperature for 2 h. The mixture
was concentrated under reduced pressure and purified by flash column chromatography on
silica gel (EtOAc/PE = 1/7) to give give the compound 1 (TMPA) (90 mg, yield: 80%) as a
colorless needle crystal (purity: > 99.99%). Mp 45-52 C (EtOAc/PE); IR (film) �max: 3437,
2931, 2856, 1737, 1681, 1494, 1454, 1403, 1334, 1163, 1138, 1117, 1030 cm-1; 1H NMR (400
MHz, CDCl3) � 0.70-0.99 (m, 3H, CH3), 1.01-1.50 (m, 11H, (CH2)4, CH3), 1.50-1.85 (m, 2H,
CH3CH2), 2.75-2.95 (t, J = 7.6 Hz, 2H, COCH2), 3.68-3.98 (m, 11H, 3OCH3, ArCH2),
4.00-4.23 (q, J = 7.2 Hz, 2H, OCH2CH3), 7.05 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) �
14.1 14.3, 22.6, 24.4, 29.1, 29.2, 31.7, 31.9, 41.0, 56.2, 60.6, 60.8, 61.1, 108.4, 121.8, 133.6,
145.1, 151.8, 152.8, 172.1 203.1; MS (ESI) m/z 403 (M+Na+, 100%). Anal. Calcd. for
C21H32O6: C, 66.29; H, 8.48. Found: C, 66.16; H, 8.72.
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References 1. McCoy,A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674 (2007).
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3. Murshudov,G.N., Vagin,A.A., & Dodson,E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. Biol. Crystallogr. 53, 240-255 (1997).
4. Emsley,P., Lohkamp,B., Scott,W.G., & Cowtan,K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486-501 (2010).
5. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D. Biol. Crystallogr. 50, 760-763 (1994).
6. Furumoto,Y. et al. Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival. J. Immunol. 176, 5167-5171 (2006).
7. Chen,Z.P. et al. Pure and functionally homogeneous recombinant retinoid X receptor. J. Biol. Chem. 269, 25770-25776 (1994).
8. Cogan,U., Kopelman,M., Mokady,S., & Shinitzky,M. Binding affinities of retinol and related compounds to retinol binding proteins. Eur. J. Biochem. 65, 71-78 (1976).
9. Zhao,B.X. et al. p53 mediates the negative regulation of MDM2 by orphan receptor TR3. EMBO J. 25, 5703-5715 (2006).
10. Ganley,I.G. et al. ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J. Biol. Chem. 284, 12297-12305 (2009).
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a
Supplementary Figure 1: (a) Nur77 is unable to affect the formation of AMPK complexes. HA-AMPK / , Flag-AMPK , and increasing amounts of Myc-Nur77 as indicated were cotransfected into 293T cells. Cell lysates were prepared and Flag-AMPK was immunoprecipitated with anti-Flag antibody followed by western blot using anti-Myc and anti-HA antibodies. (b) Determination of endogenous and exogenous Nurr77-LKB1 interactions in different cell lines. For endogenous detection, cell lysates from LO2, HCT116 and H1299 cells were immunoprecipitated with anti-LKB1 antibody, and anti-Nur77 antibody was used to determine endogenous Nur77. For transfection, HA-Nur77 and Myc-LKB1 were transfected into 293T cells. Cell lysates were immunoprecipitated with anti-HA antibody and analyzed by western blot using anti-Myc antibody. For the in vitro pull-down assay, the purified bead-bound GST or GST-LKB1 proteins was incubated with His-Nur77, and then analyzed by western blot with anti-Nur77 antibody. (c) LKB1 interacts with Nur77 but not RXR�. Different plasmids as indicated were transfected into 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed by western blot using anti-Myc antibody to indicate the association of LKB1 with Nur77 but not with retinoid X receptor (RXR ).
c
Supplementary Results
b
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dd
ee
Supplementary Figure 1 continued: (d) LKB1 binds to the ligand-binding domain (LBD) of Nur77. Left, schematic diagrams illustrate the different Nur77 truncation mutants. Right, Myc-LKB1 and different HA-Nur77 truncation mutants as indicated were co-expressed in 293T cells. Cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by western blot using anti-HA antibody. (e) Ligand binding domain (LBD) of Nur77, but not the DNA binding domain (DBD) or the transactivation domain (TAD), is necessary and sufficient to abolish the AMPK -LKB1 interaction. Both full-length Nur77 and LBD were able to affect LKB1/AMPK (1-312) complex in 293T cells, whereas neither TAD nor DBD was, in co-IP assays. The levels of AMPK phosphorylation (p-AMPK ) is indicated in the bottom panel.
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aa bb
Supplementary Figure 2: (a) TMPA increases the phosphorylation levels of AMPK via Nur77 mediation. Endogenous Nur77 in LO2 cells was knocked down by lentivirus-based RNA interference and the cells were treated with TMPA at 10 M for 6 hours. AMPKphosphorylation was detected by western blot. (b) The overlap of UV absorption spectrum of TMPA with the fluorescence emission spectrum of Nur77(LBD). a, the UV absorption spectrum of TMPA (25 M); b, the fluorescence emission spectrum of His-Nur77(LBD) (5 M). (d)TMPA cannot induce Nur77 transactivation activity. LO2 cells were transfected with a reporter gene (Nur77 response element, NurRE), then treated with TMPA (0.5 M) for the times indicated. The luciferase assay was carried out. Results are presented as the means SEM. Representatives of three independent experiments with similar results are shown. NS: non-significant vs control.
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Supplementary Figure 2 continued: (c) Fluorescence spectra of His-Nur77 (5 M), His-LBD (5 M), GST-LKB1 (5 M), His-AMPK subunits ( , and subunits, 2 M each), and AMPK / / hetorotrimer (5 M) in the absence or increasing amounts of TMPA as indicated. GST was used as the inner filter controls. TMPA binds to Nur77 and LBD but not to any of the AMPK subunits ( , and ), AMPK / / heterotrimer, or LKB1. Kd is presented as means SEM of three independent reactions. R2 is coefficient of determination. Right, all the proteins mentioned above were expressed with correct size and good purity.
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bb
a
Supplementary Figure 3: (a) Superimposition of a previous structure (2QW4 in yellow) and the asymmetric unit of LBD. Molecule-II (pink) could be superposed with 2QW4 well while molecule-I (blue) was with changes in several helices particularly in H9 and H10. (b)Superimposition of apo LBD (gray) and TMPA-bound LBD (blue). TMPA molecules are drawn as sticks in yellow and pink. The conformations between apo and TMPA-bound protein were essentially the same.
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Molecule I (site A) Molecule I (site B)
Supplementary Figure 3 continued: (c) The 2Fo-Fc electron density omit maps contoured at 1.0 and 0.7 respectively for TMPA molecules in site A (yellow) and site B (pink). Water molecules are drawn as red spheres. Hydrogen bonds are drawn as dotted lines in gray. Residues within 6 Å of TMPA are shown in sticks.
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aa
cc
Supplementary Figure 4: (a) Left, conformation analysis of Nur77(LBD) and its mutants (1 M) by circular dichroism spectroscopy, which indicated that the secondary structures were not
perturbed by the mutations. Right, Nur77(LBD) and its mutant proteins were expressed with correct size and purified to high quality. (b) The mutants displayed similar unfolding responses to the native protein �3 M each). Left, the intrinsic Trp fluorescence for both the native and mutant proteins was quenched in a similar fashion in response to the addition of urea. Right, the quenching of the Fluorescence is accompanied by a red shift. Both indicated that the mutant displayed similar unfolding response to the native protein. (c) Fluorescence of spectra of His-LBD(B3), LBD(A2) and LBD(A2/B3) (5 M each) in the absence or increasing amounts of TMPA as indicated. GST was used as an inner filter control. TMPA binds to LBD(B3) and LBD(A2) but not to LBD(A2/B3). Kd is represented as means SEM of three independent reactions. R2 is coefficient of determination.
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Supplementary Figure 4 continued: (d) Binding kinetic analysis of GST-LKB1 to His-LBD or its mutants by bio-layer interferometry. Biotinylated His-LBD or its mutants was immobilized to streptavidin-coated biosensors, and the binding of GST-LKB1 was assessed at the indicated concentrations (1.778, 0.444, 0.222, 0.111, and 0.0556 M). The binding affinity is calculated using 1:1 model and the average Kd is extracted from binding results for five different concentrations of the analytes using OctetRed system. R2 is the coefficient of determination.
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ee
Supplementary Figure 4 continued: (e) TMPA does not bind to Nur77(C566R/B3). The fluorescence spectra were measured for Nur77(C566/B3) (5 M) with increasing amounts of TMPA. (f) Overall structure and the LBD-LKB1 interactions in molecule-I. Ribbon diagram of the heterodimeric complex are shown with two TMPA molecules in yellow sticks and LKB1 in red, LBD in blue and pink ribbons. LKB1 binding to LBD in the absence (left) or presence of TMPA (right).
ff
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aa bb
dd
Supplementary Figure 5: (a) The subcellular locations of AMPK , LKB1 and Nur77 in LO2 cells and primary hepatic cells (PHC) separated from the livers of WT mice. The cytoplasmic (C) and nuclear (N) fractions were prepared, and western blot was performed to determine the protein levels of Nur77, LKB1, and AMPK .(b) Nur77 is not a phosphorylation substrate for LKB1 as determined by an in vitro kinase assay. His-Nur77 was incubated with LKB1 that was immunoprecipitated from 293T cells for the analysis by western blot with autoradiography. The phosphorylation of AMPK (1-312) by LKB1 was used as the positive control. (d) TMPA treatment (10 M, 6 hours) inhibits Nur77-LKB1 interaction in the nucleus while enhances LKB1-AMPK interaction in the cytoplasm of LO2 cells. Nuclear (N) and cytoplasmic (C) fractions of LO2 cells were prepared and immunoprecipitated with anti-LKB1 antibody, the anti-Nur77 or anti-AMPK antibody was used to determine endogenous Nur77 or AMPK .
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Supplementary Figure 5 continued: (c) TMPA treatment leads to more LKB1 translocation to the cytoplasm. HeLa cells or LO2 cells were transfected with Flag-LKB1 or LKB1(DNLS) (top), or co-transfected Flag-LKB1 or LKB1(S428A) along with cherry-Nur77 (bottom) as indicated. After transfection, cells were treated with TMPA (10 M, 6 hours) for the immunofluorescent staining. The localization of Nur77, LKB1 and its mutants were observed under confocal microscope. Scale bar: 10 m.
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ff
ee
Supplementary Figure 5 continued: (e) Nur77 inhibits (left) but TMPA increases (right) the interactions between LKB1 with STRAD and Mo25. HA-STRAD , HA-MO25, Flag-LKB1 and Myc-Nur77 were cotransfected into 293T cells as indicated, and treated with TMPA (10 M, 6 hours). Co-IP assays were performed. (f) Effect of TMPA on MARK phosphorylation in LO2 cells. Cells were treated with TMPA (10 M, 6 hours). The level of MARK phosphorylationwere determined by western blot with an antibody specific to the phosphoryated T172 of MARK and quantified by densitometry. AMPK was used as a positive control. Basal level of MARK and AMPK phosphorylation were normalized to unity. Data are presented as the means SEM from three independent experiments. Differences between groups were assessed by a two-tailed unpaired student’s t-test using SPSS software. * p < 0.05, ** p < 0.01, NS non-significant vs control.
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c d
f
aa
e
Supplementary Figure 6: (a-b) TMPA increases the phosphorylation levels of AMPK and suppresses the gluconeogenesis genes G6pase and Pepck in male db/db mice (n=8), as determined by western blot and real-time PCR. Protein and RNA were prepared from the livers of eight mice. (c-d) TMPA increases the phosphorylation of AMPK and suppresses the gluconeogenesis genes G6pase and Pepck in diabetic Nur77+/+ but not Nur77-/- db/db mice, as determined by western blot and real-time PCR. Protein and RNA were prepared from the livers of the mice (n=6 for each group). (e-f) TMPA increases the phosphorylation of AMPK and suppresses the gluconeogenesis genes G6pase and Pepck in HFD/STZ-induced diabetic Nur77+/+ mice but not in the corresponding Nur77-/- diabetic mice, as determined by western blot and real-time PCR. Protein and RNA were prepared from the mouse livers (n=8 for each group). All results above are presented as the means SEM. Representatives of two independent experiments with similar results are shown. * p < 0.05, ** p < 0.01 vs. control.
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Supplementary Table 1
Mutants from the full-length Nur77 and LBD
Site A Site B
Mutants C566R T595E H372W H494W K456S
LBD(A2)LBD(B3)LBD(A2/B3)LBD(C566R/B3) LBD(T595E/B3) Nur77(C566R/B3) Nur77(T595E/B3)
+-++-+-
+-+-+-+
-++++++
-++++++
-++++++
Mutants LBD(A2), LBD(B3) and LBD(A2/B3) were with mutations in the ligandbinding domain.
Mutants LBD(C566R/B3) and LBD(T595E/B3) ) were generated on thescaffold of LBD(B3).
Mutants Nur77(C566R/B3) and Nur77(T595E/B3) ) were mutants of the full-length Nur77.
C566R: Cys566 to Arg;T595E: Thr595 to Glu;H372W: His372 to Trp;H494W: His494 to Trp;K456S: Lys456 to Ser.
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Supplementary Table 2
Statistics in the structural characterization of Nur77 and its complex with TMPA
apo Nur77 LBD TMPA-bounded Nur77 LBD
data collection
wavelength(Å) 1.5418 1.5418
space group P212121 P212121
cell dimensionsa, b, c(Å)
, , ( )
74.43, 76,77, 128.9590.0, 90.0, 90.0
74.24, 76.77, 129.1990.0, 90.0, 90.0
resolution(Å) 2.06 2.22
Rsym or Rmergea 0.056 0.070
I/ I 10.1 7.0
completeness(%) 92.5 93.6
refinement
resolution(Å) 2.06 2.22
No. reflections 40801 33113
Rwork/Rfreeb 0.1952/0.2650 0.2092/0.2862
No. atomsproteinligandglycerolwater
37050
18480
37675424
414
R.m.s deviations bond lengths(Å)bond angles( )
0.01882.1014
0.01551.937
Mean B value(Å2) 32.425 35.76
ramachandran analysis
favored region(%) 97.59 96.53
allowed region(%) 2.19 3.04
outliers(%) 0.22 0.43
a Rmerge = h i(|Ii(h)| � |<I(h)>|)/ h iIi(h), where Ii(h) is the observed intensity, and <I(h)> is the mean intensity observed from multiple measurements.b Rwork/free = |Fobs � Fcalc|/ |Fobs|. Fobs and Fcalc are observed and calculated structure factors, Rfreeis calculated from a randomly chosen 5% of reflections (2 ), and Rwork is calculated for the remaining 92.5% of reflections (2 ).r.m.s.d is the root-mean square deviation from ideal geometry.
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Nature Chemir.m gys.d is th me root-mean square deviation
Supplementary Table 3
Small molecule screening data
Category Parameter Description
Assay Type of assay Cell-based Target AMPK acitivity
Primary measurement Detection of AMPK� phosphorylation levels Key reagents Anti-phospho- AMPK�(T172)� antibody (Cell
Signaling Technology) Assay protocol Western blot
Additional comments None
Library Library size 106 compounds
Library composition a series of compounds derived from Nur77 agonist Cytosporone B
Source synthesized Additional comments None
Screen Format Western blot
Concentration(s) tested 10 M Plate controls DMSO (0.02%) Reagent/ compound dispensing system Compound was dissolved in DMSO
Detection instrument and software Photoshop CS3 Assay validation/QC standard deviation of controls Correction factors None
Normalization The ratio of p-AMPK� and AMPK� was normalized to that of the negative control (vehicle: DMSO)
Additional comments None
Post-HTS analysis Hit criteria > 2 folds Hit rate 0.94%
Additional assay(s) None
Confirmation of hit purity and structure NMR Additional comments None
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Supplementary Figure 7: full scans of original blots for data in figure 1, 2, 3, 4, 5, 6. Panels corresponding to the figures in the paper are indicated.
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Supplementary Figure 7 continued.
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Supplementary Figure 7 continued.
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Supplementary Figure 7 continued.
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Supplementary Figure 7 continued.
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