In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl

Subscriber access provided by Iowa State University | Library

Ecotoxicology and Human Environmental Health

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl Substances to Baikal Seal and Human Peroxisome Proliferator-Activated Receptor # Hiroshi Ishibashi, Hirano Masashi, Eun-Young Kim, and Hisato Iwata Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07273 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Environmental Science & Technology

1

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl

2

Substances to Baikal Seal and Human Peroxisome Proliferator-Activated

3

Receptor α

4 Hiroshi Ishibashi†,‡, Hirano Masashi§, Eun-Young Kimǁ, Hisato Iwata†,*

5 6 7

†

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,

8 9

Matsuyama 790-8577, Japan ‡ Graduate

School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566,

10

Japan

11

§ Department

12

Technology, Kumamoto College, 2627 Hirayama-shinmachi, Yatsushiro, Kumamoto

13

866-8501, Japan

14 15

ǁ Department

of Biological and Chemical Systems Engineering, National Institute of

of Life and Nanopharmaceutical Science and Department of Biology, Kyung

Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea

16 17

* Address

correspondence to:

1 ACS Paragon Plus Environment


18

Hisato Iwata,

19

Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5,

20

Matsuyama 790-8577, Japan.

21

Tel/Fax: +81-89-927-8172

22

E-mail: [email protected]

23


Page 2 of 38

Page 3 of 38


24

ABSTRACT: In this study, we assessed the binding affinities of perfluoroalkyl substances

25

(PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates

26

(PFSAs), to the ligand-binding domains (LBDs) of Baikal seal (Pusa sibirica; bs) and human

27

(h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive

28

binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and

29

hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs)

30

of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA >

31

PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation

32

with those to hPPARα. In silico PPARα homology modeling predicted that there were two

33

ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity

34

relationship analyses suggested that the binding potencies of PFASs to PPARα might depend

35

on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated

36

carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding

37

affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains

38

than that of hPPARα. The in silico docking simulations suggested that the 1st LBP of

39

bsPPARα had higher affinities than that of hPPARα; however, the 2nd LBP of bsPPARα had

40

lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing



41

interspecies differences in the binding of PFASs to PPARαs and their structure-activity

42

relationships.

43 44

INTRODUCTION

45

Perfluoroalkyl substances (PFASs), such as perfluoroalkyl carboxylates (PFCAs) and

46

perfluoroalkyl sulfonates (PFSAs), with different carbon chain length are man-made

47

chemicals, which have been globally detected in the environment, humans and wildlife.1–8

48

Owing to their high environmental persistence, bioaccumulation potential, and toxic

49

properties, perfluorooctane sulfonate (PFOS) and its salts, as well as perfluorooctane sulfonyl

50

fluoride have been globally regulated by the Stockholm Convention on Persistent Organic

51

Pollutants.9,10 However, the toxic effects and risks of different PFASs in animals, particularly

52

the non-model wildlife, are not fully understood.

53

Previous studies have shown that PFASs, including PFOS and perfluorooctanoic acid

54

(PFOA), exert a wide range of toxic effects, including reproductive and developmental

55

toxicity, hepatotoxicity, carcinogenesis, neurotoxicity, immunotoxicity, and hormonal effects,

56

in experimental animals.8,11–13 The results of these studies have suggested that adverse effects

57

of PFASs are mostly mediated by peroxisome proliferator-activated receptors (PPARs).

58

PPARs are members of the ligand-activated nuclear receptor superfamily, and three different 4 ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38


59

PPAR subtypes, PPARα, PPARβ/δ, and PPARγ, have been identified in various animal

60

species.14–16 The ligand-PPAR complex heterodimerizes with retinoid X receptor α (RXRα),

61

interacts with the peroxisome proliferator-responsive element (PPRE) in the promoter regions

62

of target genes, such as cytochrome P450 (CYP) 4A, and consequently regulates their

63

transcriptional activity.11 Studies in PPARα gene knockout mice clearly showed that PPARα

64

regulated the expression of CYP4A, which can catalyze the oxidation of fatty acids.17,18

65

Exposure to PFOA, as well as fatty acids, can also upregulate hepatic CYP4A mRNA and

66

protein expression in rats.19 Previous studies using in vitro reporter gene assays have

67

indicated differences in the transactivation potencies of PFASs with different carbon chain

68

length, including PFOS and PFOA, for human, mouse, and rat PPARα,20–23 suggesting

69

significant interspecies differences among PPARα in their ligand preference. However,

70

limited data are available on the interspecies differences in PPARα activation by PFASs.

71

The Baikal seal (Pusa sibirica), a top predator in Lake Baikal, Russia, accumulates

72

high levels of environmental pollutants.24–26 Our research group has previously determined

73

the residual levels of various PFASs in the liver and serum of wild Baikal seals, which were

74

particularly high for PFOS, perfluorononanoic acid (PFNA), and perfluorodecanoic acid

75

(PFDA).27 We have further obtained and sequenced the full-length cDNA of Baikal seal



76

PPARα (bsPPARα), showing that PPARα mRNA and a CYP4A-like protein expression were

77

upregulated by high levels of PFASs, such as PFNA and PFDA in the liver of wild seals.28

78

Our in vitro reporter gene assay, using a bsPPARα expression vector and a PPRE-containing

79

reporter vector, has shown that certain PFASs, such as PFOS, PFOA, PFNA, PFDA, and

80

PFUnDA, had transactivation capabilities for bsPPARα.29 These results indicated that

81

bsPPARα-mediated response might be useful for assessing potential biological/toxicological

82

effects of PFASs on the wild Baikal seal population. However, it has not been fully

83

investigated whether a molecular event, such as the binding of PFASs to PPARα triggers its

84

downstream signaling. Although various studies have reported the in vitro transactivation

85

potencies of certain PFASs for PPARα,20–23 it is not known yet whether any of these

86

chemicals directly interacts with bsPPARα. Additionally, a previous study identified another

87

new ligand-binding pocket (2nd LBP) in the crystal structure of human PPARα (hPPARα),

88

besides the classical LBP of hPPARα, 1st LBP.30 However, the binding potencies of PFASs to

89

these two LBPs of PPARα remain unclear.

90

In this study, we aimed to evaluate the direct binding affinities of PFCAs and PFSAs

91

with various carbon chain lengths (C4-C11) to bsPPARα and hPPARα, as well as their

92

structure-activity relationships and interspecies differences. We expressed and purified a


Page 6 of 38

Page 7 of 38


93

recombinant bsPPARα ligand-binding domain (LBD) protein using an in vitro wheat germ

94

cell-free protein synthesis system and measured the direct binding affinities of PFASs to

95

bsPPARα and hPPARα LBDs using a competitive binding assay. We further performed in

96

silico docking simulation analyses to predict the molecular interactions of PFASs with the 1st

97

and 2nd LBPs of bsPPARα and hPPARα LBDs. Finally, we compared the structure-activity

98

relationships among PFASs with respect to their binding to bsPPARα and hPPARα LBDs

99

and evaluated the interspecies differences.

100 101 102

MATERIALS AND METHODS Chemicals. Eleven PFASs (eight PFCAs and three PFSAs) were used as test

103

compounds, and their full names, providers, and purities are shown in the Supporting

104

Information (Table S1). A PPARα agonist,

105

[4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphen

106

oxy]acetic acid (GW0742),31,32 used as a positive control ligand, was obtained from Cayman

107

Chemical (Ann Arbor, MI, USA). For all PFASs, 250 mM stock solutions were prepared by

108

dissolving each chemical in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO,

109

USA), and the final DMSO concentration was adjusted to 1% (v/v). A 1.85 mM stock



110

solution of GW0742 was prepared in a similar manner. For the PPARα competitive binding

111

assay, test solutions of PFASs (final concentrations: 1.14-2,500 µM) and GW0742

112

(0.31-18,500 nM) were prepared by serially diluting the stock solutions with the binding

113

assay buffer included in the PPARα binding assay kit (Invitrogen, Tokyo, Japan).

114

Plasmid Construction and Recombinant Protein Synthesis. The bsPPARα LBD

115

cDNA (National Center for Biotechnology Information accession number: LC072268) was

116

amplified by polymerase chain reaction using specific primers, containing XhoI and NotI

117

restriction enzyme recognition sites. The amplicon of bsPPARα LBD cDNA was then

118

digested with the restriction enzymes and subcloned into the XhoI/NotI site of the

119

pEU-E01-GST-TEV-MCS-N2 expression vector (CellFree Sciences Co., Ltd., Ehime,

120

Japan), and the subcloned bsPPARα LBD cDNA was sequenced to ensure that no mutations

121

were introduced by PCR, as previously reported.28 bsPPARα LBD mRNA was prepared by in

122

vitro transcription using SP6 RNA polymerase. A glutathione-S-transferase (GST)-tagged

123

bsPPARα LBD protein was synthesized using an in vitro wheat germ cell-free protein

124

synthesis system, with some modifications.33 The GST-tagged bsPPARα LBD protein was

125

purified on glutathione Sepharose™ 4B (GE Healthcare, Tokyo, Japan), according to the

126

manufacturer’s instructions, and the purified yield was confirmed by sodium dodecyl sulfate


Page 8 of 38

Page 9 of 38


127

polyacrylamide gel electrophoresis (SDS-PAGE). A recombinant hPPARα LBD protein was

128

purchased from Invitrogen.

129

PPARα Competitive Binding Assay. The competitive binding of the test chemicals to

130

bsPPARα and hPPARα LBD proteins was determined using the LanthaScreen™

131

time-resolved fluorescence resonance energy transfer (TR-FRET) PPARα competitive

132

binding assay kit, according to the manufacturer's instructions (Invitrogen). Briefly, serially

133

diluted solutions of the test chemicals (10 µL) were prepared in a 384-well polypropylene

134

assay plate. Then, 5 µL of 4× Fluormone™ Pan-PPAR Green was added into each well,

135

followed by the addition of 5 µL of 4× GST-tagged PPARα LBD protein/terbium-labeled

136

anti-GST antibody in the TR-FRET assay buffer. The plate was gently mixed and then

137

incubated at room temperature for 2 h. TR-FRET was measured at emission wavelengths of

138

520 and 495 nm using an Infinite™ F500 fluorescence plate reader (Tecan Japan Co., Ltd.,

139

Kanagawa, Japan) with the recommended settings. The results are presented as the means ±

140

standard deviations from three independent experiments, in which each consisted of two

141

technical replicates for each concentration tested. A binding curve was generated by plotting

142

the 520/495 nm ratio versus the log concentration, and the median inhibitory concentration

143

(IC50) was determined as the competitor concentration that caused 50% displacement of



144

Fluormone™ Pan-PPAR Green using GraphPad Prism™ version 7.0 (GraphPad Software,

145

San Diego, CA, USA). Regarding PFBA, PFPeA, and PFBS, which showed weak binding

146

potencies to both PPARαs, a binding curve over 50% inhibition was not obtained; thus, the

147

IC50 value for each chemical was predicted from a regression line using plots from the

148

highest three test concentrations. The relative binding affinity (RBA) of PFASs to bsPPARα

149

and hPPARα LBDs was estimated as the ratio of IC50 of PFOA to that of the test chemicals,

150

and PFOA was selected as a reference to compare the RBAs among PFASs.

151

Homology Modeling and Docking Simulation. Homology modeling of bsPPARα

152

LBD protein and docking simulation analysis were performed using the Molecular Operation

153

Environment (MOE) program (Chemical Computing Group, Inc., Montreal, QB, Canada), as

154

previously described.34,35 The homology model of bsPPARα LBD was constructed based on

155

the X-ray crystal structure of the hPPARα LBD bound with WY14643 (Protein Data Bank

156

ID: 4BCR), because both of 1st and 2nd LBPs were identified only in this structure.30 Detailed

157

procedures are described in the Supporting Information.

158 159 160

RESULTS AND DISCUSSION Expression of bsPPARα LBD Protein and Binding Affinity of GW0742. An X-ray


Page 10 of 38

Page 11 of 38


161

crystallographic structure analysis and in vitro binding assays have shown the utility of

162

human and rodent recombinant PPARα LBD proteins expressed in Escherichia coli.36,37

163

However, the in vitro wheat germ cell-free protein synthesis technology, developed by Endo

164

and Sawasaki,33,38 is advantageous in terms of the quality and quantity of various synthesized

165

recombinant proteins. In the present study, a GST-tagged bsPPARα LBD protein was

166

synthesized using the in vitro wheat germ cell-free protein synthesis system. SDS-PAGE of

167

the purified protein showed a single band of bsPPARα LBD of the expected molecular

168

weight (approximately 52 kDa) (Figure S1, lane 4). This result suggested that recombinant

169

GST-tagged bsPPARα LBD protein was successfully synthesized using this in vitro system.

170

Prior to investigating the binding potential of PFASs to bsPPARα LBD, the binding

171

affinity of GW0742, a PPARα agonist, was measured using the LanthaScreen™ TR-FRET

172

assay system. In this assay, ligand binding to PPARα LBD was assessed by displacement of a

173

fluorescent PPARα ligand (Fluormone™ Pan-PPAR Green) from the receptor, leading to a

174

decrease in the FRET signal derived from the interaction of the fluorescent ligand and a

175

terbium-labeled antibody with the GST-tag on PPARα.39 Results showed that GW0742

176

displaced Fluormone™ Pan-PPAR Green from bsPPARα LBD in a concentration- and

177

time-dependent manner (Figure S2A). The IC50 values of GW0742 were 217 nM (95%



178

confidence interval: 112-421 nM) at 0.5 h, 260 nM (131-515 nM) at 1 h, 339 nM (162-709

179

nM) at 2 h, and 371 nM (168-818 nM) at 3 h. Similarly, GW0742 also showed a binding

180

affinity to hPPARα LBD in a concentration- and time-dependent manner (IC50 values: 140,

181

184, 207, and 221 nM at 0.5, 1, 2, and 3 h, respectively; Figure S2B), and these values were

182

lower than the IC50 value (680 nM) of GW0742.39 Comparison of the IC50 values of GW0742

183

for bsPPARα and hPPARα LBDs showed that they were similar at 2 and 3 h, suggesting that

184

the reactions reached a steady state after 2-h exposure. These results suggested that both

185

bsPPARα LBD, synthesized in vitro in this study, and hPPARα LBD proteins were functional

186

and able to bind to the PPARα agonist, GW0742.

187

Competitive Binding of PFASs to bsPPARα and hPPARα. Several previous studies

188

have shown that PFOS and PFOA can directly bind to a liver fatty acid-binding protein and

189

serum binding protein in several animal species.40–48 However, to our knowledge, there are

190

limited data on the direct binding of PFASs to the PPARα protein, although a recent study

191

using an in vitro binding assay reported the direct binding of PFOS to hPPARα LBD

192

protein.40 Thus, in the present study, we assessed the direct binding abilities of PFCAs and

193

PFSAs with various numbers of perfluorinated carbons for bsPPARα and hPPARα LBDs

194

using the LanthaScreen™ TR-FRET assay. Among the 11 PFASs tested, six PFCAs


Page 12 of 38

Page 13 of 38


195

(PFHxA, PFHpA, PFOA, PFNA, PFDA, and PFUnDA) and two PFSAs (PFHxS and PFOS)

196

could directly bind to bsPPARα (Figures S3 and S4) and hPPARα LBDs (Figures S5 and S6)

197

in a dose-dependent manner. For hPPARα LBD, the IC50 value of PFOS was 237 µM (Table

198

1), which is in line with the results of a previous study (IC50: 247.7 µM).40 However, PFASs

199

with shorter carbon chains, such as PFBA (number of perfluorinated carbons: 3), PFPeA (4),

200

and PFBS (4), exhibited weak binding responses (˂ 50% inhibition) only at high

201

concentrations for both PPARα LBDs (Figures S3-S6, A and/or B). To our knowledge, this is

202

the first evidence on the direct binding of PFCAs and PFSAs with various numbers of

203

perfluorinated carbons to bsPPARα and hPPARα LBDs.

204

Structure-Activity Relationships of PFASs and Their Binding to bsPPARα and

205

hPPARα. An in vitro competitive binding assay was employed to determine the binding

206

affinities of short/long-chain fatty acids to hPPARα LBD and their structure-activity

207

relationships.49,50 However, the relationship between the structure of short/long-chain PFASs

208

and their PPARα-binding affinities has not been investigated yet. To compare the

209

bsPPARα-binding potencies of PFASs, we initially calculated the RBA of each PFAS to

210

bsPPARα LBD relative to that of PFOA (Table 1). PFOS showed the highest binding affinity

211

to bsPPARα LBD. The order of RBAs for the 11 PFASs was as follows: PFOS (3.75) >



212

PFDA (2.24) > PFNA (1.71) > PFUnDA (1.36) > PFOA (1) > PFHxS (0.65) > PFHpA (0.53)

213

> PFHxA (0.11) >> PFBA (0.067), PFPeA (0.10), and PFBS (0.043) (Table 1). Our previous

214

study has suggested, based on the data obtained from an in vitro reporter gene assay, that the

215

number of perfluorinated carbons in PFCAs is one of the factors determining their

216

transactivation potencies for bsPPARα.29 Therefore, the current study evaluated the

217

relationship between the number of perfluorinated carbons in PFASs and their RBAs.

218

Pearson correlation test revealed a positive correlation (p = 0.0001, r = 0.818) between the

219

number of perfluorinated carbons and RBAs of PFASs with 3-10 perfluorinated carbons to

220

bsPPARα LBD (Figure 1A). For hPPARα LBD, the RBAs of PFASs also increased with the

221

increase in the number of perfluorinated carbons (p = 0.0072, Figure 1B), although the RBA

222

of PFUnDA (number of perfluorinated carbons: 10) appeared to be an outlier. These results

223

suggested that the number of perfluorinated carbons in PFASs, besides the fatty acid chain

224

length, might contribute to their binding potencies for bsPPARα and hPPARα LBDs.49

225

On the other hand, our previous bsPPARα transactivation assay has indicated that

226

PFCAs with more than seven perfluorinated carbons showed a negative correlation with

227

PFOA induction equivalent factors (IEFs).29 Comparison of RBAs and IEFs of PFASs that

228

were obtained from the bsPPARα-binding and transactivation assays, respectively, revealed


Page 14 of 38

Page 15 of 38


229

that IEFs were lower than RBAs for PFASs with more than eight perfluorinated carbons,

230

such as PFNA (IEF: 0.61), PFDA (0.37), PFUnDA (0.15), and PFOS (0.26).29 The data from

231

the present and previous studies suggest that bsPPARα is activated through direct binding of

232

PFASs. However, the high binding affinity but low transactivation potency of PFNA, PFDA,

233

PFUnDA, and PFOS suggest that these compounds may be partial antagonists for bsPPARα

234

transactivation. Further study is necessary to assess the antagonist potencies of these PFASs

235

for bsPPARα transactivation.

236 237

Table 1. In Vitro Binding Potencies of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkyl

238

Sulfonates (PFSAs) for the Baikal Seal and Human PPARα Ligand-Binding Domains and

239

Interspecies Differences

IC50 (µM)a

RBAb

abbreviation

IC50 ratioc bsPPARα

hPPARα

bsPPARα

hPPARα

PFCAs

PFBA

4,637

3,224

0.067

0.12

1.44

PFPeA

3,036

3,279

0.10

0.11

0.93

PFHxA

2,790 ± 1.31

904 ± 2.13

0.11

0.41

3.08



Page 16 of 38

PFHpA

586 ± 1.14

275 ± 1.54

0.53

1.35

2.13

PFOA

311 ± 1.40

371 ± 1.62

1

1

0.84

PFNA

182 ± 2.16

277 ± 2.48

1.71

1.34

0.66

PFDA

139 ± 2.41

366 ± 4.57

2.24

1.01

0.38

PFUnDA

228 ± 2.85

3,265 ± 6.38

1.36

0.11

0.070

7,231

7,745

0.043

0.048

0.93

PFHxS

480 ± 1.55

140 ± 1.71

0.65

2.65

3.44

PFOS

83 ± 2.18

237 ± 2.55

3.75

1.57

0.35

PFSAs

PFBS

240

aMedian

inhibitory concentration ± standard error: the competitor concentration that causes 50%

241

displacement of FluormoneTM Pan-PPAR Green. bRelative binding affinity: IC50 of PFOA/IC50 of

242

competitor. cRatio of IC50 for the binding of bsPPARα LBD to IC50 for the binding of hPPARα LBD.

243

The IC50 values for PFBA, PFPeA, and PFBS were predicted from a regression line using plots from

244

the highest three test concentrations, because these chemicals showed weak binding potencies to both

245

PPARαs and a binding curve over 50% inhibition was not obtained.

246


Page 17 of 38


2

A

y = 0.271x − 2.06 p = 0.0001, r = 0.818

B

y = 0.233x − 1.67 p = 0.0072, r = 0.615

Log [relative binding affinity (RBA)]

1 0 -1 -2 2 1

PFBA (3) PFPeA (4) PFHxA (5) PFHpA (6) PFOA (7) PFNA (8) PFDA (9) PFUnDA (10) PFUdA (10) PFBS (4) PFBuS (4) PFHxS (6) PFOS (8)

0 -1 -2

3

4

5

6

7

8

9

10

247

Perfluorinated carbon number

248

Figure 1. Relationships between the numbers of perfluorinated carbons in perfluoroalkyl

249

substances (PFASs) and their relative binding affinities (RBAs) to the (A) Baikal seal PPARα

250

ligand-binding domain (LBD) and (B) human PPARα LBD. The number in parentheses next

251

to the PFAS name represents the respective number of perfluorinated carbons in the

252

molecule.

253 254

The present study also estimated the RBAs of PFASs to hPPARα LBD (Table 1).

255

Results showed that PFHxS had the highest RBA value (2.65) for hPPARα LBD, followed by

256

PFOS (1.57), PFHpA (1.35), and PFNA (1.34). Similarly, the hPPARα LBD-binding abilities

257

of PFASs also increased with the increase in the number of perfluorinated carbons, except for 17 ACS Paragon Plus Environment


258

PFUnDA (Figure 1B). Using an in vitro reporter gene assay, a previous study showed that

259

PFCAs with a medium chain length, such as PFHpA (number of perfluorinated carbons: 6)

260

and PFOA (7), had a high potential for hPPARα activation, whereas PFCAs with short or

261

long carbon chain lengths showed a low potential.20 These results suggested that the

262

hPPARα-binding and transactivation profiles of PFCAs were similar and the transactivation

263

of hPPARα by PFCAs directly reflected their binding.

264

Our previous studies have shown that in silico analysis is a useful tool to understand the

265

molecular basis of the binding of environmental chemicals to nuclear receptors.34,35

266

Moreover, a previous study reported a new ligand-binding pocket (2nd LBP) interacting with

267

WY14643, a typical hPPARα ligand in the crystal structure of hPPARα LBD.30 In this study,

268

we calculated the in silico binding scores (S-score, kcal/mol) for the interaction of each PFAS

269

with the 1st LBP, the classical LBP,30 and 2nd LBP of bsPPARα and hPPARα LBDs and then

270

examined the relationships between these scores and the number of perfluorinated carbons of

271

PFASs. To initially construct a homology model for bsPPARα LBD, we used the X-ray

272

crystal structure of hPPARα LBD (PDB code: 4BCR) as a template. Results revealed that the

273

bsPPARα LBD homology model closely resembled the X-ray crystal structure of hPPARα

274

LBD (Figure S7A), showing a low root-mean-square distance (RMSD) value (0.622 Å3).


Page 18 of 38

Page 19 of 38

275


Our in silico docking simulation analyses, using bsPPARα (Figure S8A) and hPPARα

276

(Figure S8B) 1st LBP models, showed negative correlations between the number of

277

perfluorinated carbons and S-scores of PFASs. All PFASs fitted into PPARα 1st LBPs of both

278

species (Figures S10 and S11). Moreover, the S-scores of PFHxDA (number of

279

perfluorinated carbons: 15) and PFODA (17) were relatively high and outliers from the

280

regression line (Figure S8A). Detailed evaluation of the S-scores showed that the E_refine

281

values (the sum of van der Waals interaction, electrostatic interaction, and solvation effect

282

energies) in the S-score of PFHxDA (E_refine value: 25.5 kcal/mol) and PFODA (37.8

283

kcal/mol) were much higher than those of the other 14 PFASs (-15.8 to -2.73 kcal/mol).

284

Thus, these energies might be involved in the low binding potentials of PFHxDA and

285

PFODA to bsPPARα 1st LBP. Taken together, these results suggested that the number of

286

perfluorinated carbons (i.e., molecular size) and E_refine values of PFASs might contribute

287

to their binding potencies for bsPPARα and hPPARα 1st LBPs.

288

For bsPPARα 2nd LBP, a significant negative correlation was observed between the

289

number of perfluorinated carbons and S-scores of PFASs (Figure S8C). However, most of

290

PFASs did not fit into the LBP (Figure S12), and their S-scores (-4.90 to -4.05 kcal/mol)

291

were higher than those of bsPPARα 1st LBP (-8.16 to -4.73 kcal/mol) and hPPARα 2nd LBP



292

(-6.00 to -4.39 kcal/mol) (Table S2). This might be attributable to the tight space of bsPPARα

293

2nd LBP (244.0 Å3, Figure S9C). Thus, the binding to bsPPARα 2nd LBP might be weaker

294

than that to bsPPARα 1st LBP. hPPARα 2nd LBP model also revealed a significant negative

295

correlation between the number of perfluorinated carbons and S-scores of PFASs (Figure

296

S8D). Although most of PFASs fitted into hPPARα 2nd LBP owing to its greater cavity

297

volume (511.3 Å3, Figure S9C) than that of bsPPARα 2nd LBP, long-chain PFASs, such as

298

PFTeDA (13), PFHxDA (15), and PFODA (17), did not fit into hPPARα 2nd LBP (Figures

299

S13K-S13M). These PFASs had no amino acid residues involved in hydrogen bond

300

interactions (Table S2). The low binding potencies of these long-chain PFASs may be

301

attributable to no-fitting in the cavity and no hydrogen bond formation with the amino acids

302

in hPPARα 2nd LBP.

303

We also examined the relationships between LogKow values and RBAs of PFASs (data

304

not shown). Results showed a similar relationship to that between the number of

305

perfluorinated carbons and RBAs of PFASs (Figure 1). Thus, the hydrophobicity of PFASs,

306

i.e. LogKow values, might also affect the binding capabilities of PFASs to bsPPARα and

307

hPPARα LBDs. Taken together, these results suggested that the binding potencies of PFASs

308

to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the


Page 20 of 38

Page 21 of 38

309 310


number of perfluorinated carbons, and hydrophobicity of PFASs. Species Differences in Binding of PFASs to bsPPARα and hPPARα. Several studies

311

have suggested species differences in the transactivation potencies of PFASs for PPARα

312

among rats, mice, and humans.21–23 However, no data are available on the species differences

313

in direct binding of PFASs to PPARα. To compare the binding affinities of PFASs among

314

species, this study estimated the ratio of IC50 values for binding to bsPPARα and hPPARα for

315

each PFAS (Table 1). Results revealed that the ratios for PFASs with five or six

316

perfluorinated carbons were more than 1, whereas the ratios for PFASs with more than seven

317

perfluorinated carbons were ˂ 1. These results suggested that bsPPARα showed preference

318

for PFASs with longer carbon chains, compared to that of hPPARα. We have previously

319

found that the amino-acid sequence of bsPPARα LBD was 96% identical to that of hPPARα

320

LBD.28 A previous study identified the key amino acids involved in hydrogen bond

321

interactions for hPPARα 1st and 2nd LBPs based on their X-ray crystal structure (WY14643,

322

Ser280, Tyr314, His440, and Tyr464 for hPPARα 1st LBP and Glu251, Lys266, His274, and

323

Asp453 for hPPARα 2nd LBP).30 The amino acids of hPPARα 1st and 2nd LBPs were similar

324

to those of bsPPARα 1st and 2nd LBPs (Figure S7). Therefore, these amino acids of PPARα

325

1st and 2nd LBPs did not account for the interspecies differences in the binding of PFASs.



326

Among the 11 PFASs tested in this study, the IC50 value (228 µM) of PFUnDA for

327

bsPPARα was much lower than that for hPPARα (3,265 µM) (Table 1), suggesting that

328

PFUnDA had the most striking interspecies difference in the binding to PPARα (Figure S14).

329

To understand the molecular basis of interspecies differences in the binding of PFASs to

330

PPARα, we compared the in silico binding scores (S-score) for the interaction of each PFAS

331

with bsPPARα and hPPARα 1st and 2nd LBPs. Results revealed that the S-scores of PFASs

332

for bsPPARα and hPPARα 1st LBPs were plotted to the left side from the 1:1 linear

333

regression line (y = x, Figure S15A); however, those for bsPPARα and hPPARα 2nd LBPs

334

were plotted to the right side from the 1:1 linear regression line (Figure S15B). Among the

335

S-scores of PFASs for PPARα 2nd LBPs, PFUnDA showed the most striking outlier from the

336

1:1 linear regression line (Figure S15B), indicating its stronger binding to hPPARα 2nd LBP

337

than that to bsPPARα 2nd LBP. For most of PFASs, the S-scores for hPPARα 1st LBP were

338

lower than or comparable to those for hPPARα 2nd LBP (Table S2). In contrast, the S-score

339

of PFUnDA for the 2nd LBP was the lowest among those of PFASs, and was lower than that

340

for the 1st LBP (Table S2). These results indicated that PFUnDA preferably bound to

341

hPPARα 2nd LBP. We further identified the key amino acids interacting with PFUnDA in

342

hPPARα 2nd LBP. A previous study examining the X-ray crystal structure of hPPARα


Page 22 of 38

Page 23 of 38


343

showed that key amino acids, such as Glu251, Lys266, His274, and Asp453 (Figure S7B,

344

green boxes), were involved in hydrogen bond interactions between hPPARα 2nd LBP and

345

WY14643; in addition, polar residues participating in hydrogen bonds were important for

346

stabilizing the ligand-receptor interaction.30 Our results showed that PFUnDA formed a

347

hydrogen bond with a polar residue (Thr279); however, the other PFASs mainly formed a

348

hydrogen bond with a non-polar residue (Cys275) (Table S2). Moreover, the E_refine value

349

of PFUnDA (E_refine value: -21.1 kcal/mol) was lower than those of other PFASs (-20.2 to

350

-10.0 kcal/mol). Thus, the higher binding potency of PFUnDA for hPPARα 2nd LBP might be

351

attributable to the formation of a hydrogen bond with a polar residue and the low E_refine

352

value in the 2nd LBP. Since the binding affinity in this study was measured based on the

353

competition between the tested PFASs and Fluormone™ Pan-PPAR Green, PFUnDA might

354

not have fully competed with the fluorescent probe in the 1st LBP, leading to an apparently

355

weak binding of PFUnDA to hPPARα. Taken together, these results suggested that there

356

were little interspecies differences in the relative binding potencies of PFASs with various

357

carbon chain lengths (C4-C11) for the bsPPARα and hPPARα LBD proteins (Figure S14),

358

although there might be interspecies differences in their absolute binding potencies to each

359

LBP (Figure S15).



360

We finally compared between the S-scores of PFASs determined using in silico

361

docking simulations (PPARα 1st and 2nd LBPs) and their RBAs determined using in vitro

362

PPARα binding assays. Results revealed a significant negative correlation between the

363

S-scores of PFASs for bsPPARα 1st and 2nd LBPs and their RBAs determined using in vitro

364

bsPPARα-binding assays (Figures 2A and 2C). In addition, the RBAs of PFASs determined

365

using in vitro hPPARα-binding assays also negatively correlated with the in silico S-scores of

366

PFASs (Figures 2B and 2D), although PFUnDA unexpectedly showed a low RBA value.

367

These results suggested that in silico docking simulation may be a useful tool for screening

368

potential ligands for bsPPARα and hPPARα 1st and 2nd LBPs.

369

Overall, our study provided the first evidence of interspecies differences in the binding

370

of PFASs to PPARαs and their structure-activity relationships. However, the toxicological

371

implications from the binding of PFASs to the PPARα remain unknown. In a previous study,

372

we have measured the hepatic concentrations of total 10 PFASs (14-143 ng/g wet weight) in

373

wild Baikal seals.27 For example, the hepatic concentrations of PFOS (mean 11.4 ng/g wet

374

weight) in the seals27 were much lower than the IC50 value of PFOS (83 µM = 42 µg/g) for

375

bsPPARα-binding affinity (Table 1). However, the wild Baikal seals are chronically exposed

376

to a mixture of PFASs.27 The mixture may additively and/or synergistically affect the


Page 24 of 38

Page 25 of 38


377

bsPPARα signaling pathway even at the low concentration of each PFAS, since the mixture

378

concentration including unknown ligands may reach the effective level at which bsPPARα is

379

activated.28 Exploring how the binding of PFASs to PPARα is transferred into their

380

downstream responses will be the next challenge for understanding the toxic effects of these

381

compounds.

Figure 2

382 383 2

22

A Log [relative binding affinity (RBA)]

1

00

-1

-1-1

-2

-8

-7

-6

-5

y = −0.980x − 5.34 p = 0.0032, r = 0.683

11

0

-4

-2-2 -6.5 -6.5

-6.0 -6.0

-5.5 -5.5

-5.0 -5.0

-4.5 -4.5

-4.0 -4.0

2

2

y = −2.63x − 11.6 p = 0.0117, r = 0.525

C 1

D 0

-1

-1

-4.8

-4.6

-4.4

-4.2

-4.0

-3.8

-2 -6.5

PFBA PFBA(3) (3) PFPeA PFPeA(4) (4) PFHxA PFHxA(5) (5) PFHpA PFHpA(6) (6) PFOA PFOA(7) (7) PFNA PFNA(8) (8) PFDA PFDA(9) (9) PFUnDA (10) PFUdA (10) PFBS PFBuS (4)(4) PFHxS PFHxS(6) (6) PFOS PFOS(8) (8)

y = −1.26x − 6.52 p = 0.0322, r = 0.456

1

0

-2 -5.0

384

B

y = −0.923x − 5.76 p < 0.0001, r = 0.825

-6.0

-5.5

-5.0

-4.5

-4.0

Docking simulation (S-score, kcal/mol)

385

Figure 2. Relationship between the S-scores obtained by in silico docking simulation and the

386

relative binding affinities (RBAs) of perfluoroalkyl substances (PFASs), obtained using an in

387

vitro Baikal seal (bs) PPARα- and human (h) PPARα-binding assay. (A) bsPPARα 1st ligand



388

binding pocket (LBP), (B) hPPARα 1st LBP, (C) bsPPARα 2nd LBP, and (D) hPPARα 2nd

389

LBP. The number in parentheses next to the PFAS name represents the respective number of

390

perfluorinated carbons in the molecule.

391 392 393

ACKNOWLEDGMENTS This study was supported by Grant-in-Aid for Scientific Research (S) (no. 21221004 &

394

26220103) and (B) (no. 24380179 & 16H05057) from Japan Society for the Promotion of

395

Science (JSPS), and “Joint Usage/Research Center – Leading Academia in Marine and

396

Environment Pollution Research (LaMer)” from the Ministry of Education, Culture, Sports,

397

Science and Technology (MEXT), Japan. Financial assistance was also provided in part by

398

Basic Research in ExTEND2005 (Enhanced Tack on Endocrine Disruption) from the

399

Ministry of Environment, Japan. We would like to thank Editage (www.editage.jp) for

400

English language editing.

401 402

ASSOCIATED CONTENT

403

Supporting Information

404

Table S1: PFASs used in this study. Table S2: Interaction energies and amino acids predicted


Page 26 of 38

Page 27 of 38


405

to participate in hydrogen bonding. Figure S1: Expression of the GST-tagged bsPPARα LBD

406

protein. Figure S2: Binding affinities of GW0742 to the bsPPARα and hPPARα LBDs.

407

Figure S3: Binding affinities of PFCAs to the bsPPARα LBD. Figure S4: Binding affinities

408

of PFSAs to the bsPPARα LBD. Figure S5: Binding affinities of PFCAs to the hPPARα

409

LBD. Figure S6: Binding affinities of PFSAs to the hPPARα LBD. Figure S7: Structural

410

comparison of bsPPARα and hPPARα LBDs. Figure S8: Relationships between the

411

perfluorinated carbon number of PFASs and the S-score. Figure 9S: Predicted cavity volume

412

of bsPPARα and hPPARα 1st and 2nd LBPs. Figure S10: Docking poses of PFASs to

413

bsPPARα 1st LBP. Figure S11: Docking poses of PFASs to hPPARα 1st LBP. Figure S12:

414

Docking poses of PFASs to bsPPARα 2nd LBP. Figure S13: Docking poses of PFASs to

415

hPPARα 2nd LBP. Figure S14: Relationship between the IC50 values of PFASs for the

416

binding to bsPPARα and hPPARα LBDs. Figure S15: Relationships between the S-scores of

417

the in silico docking of PFASs to bsPPARα and hPPARα. This material is available free of

418

charge via the Internet at http://pubs.acs.org.

419 420

AUTHOR INFORMATION

421

Corresponding Author



422

*Phone: +81-89-927-8172; e-mail: [email protected]

423

Notes

424

The authors declare no competing financial interest.

425 426

REFERENCES

427

(1)

428 429

Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35, 1339–1342.

(2)

Kannan, K.; Koistinen, J.; Beckmen, K.; Evans, T.; Gorzelany, J. F.; Hansen, K. J.;

430

Jones, P. D.; Helle, E.; Nyman, M.; Giesy, J. P. Accumulation of perfluorooctane

431

sulfonate in marine mammals. Environ. Sci. Technol. 2001, 35, 1593–1598.

432

(3)

Kannan, K.; Corsolini, S.; Falandysz, J.; Fillmann, G.; Kumar, K. S.; Loganathan, B.

433

G.; Mohd, M. A.; Olivero, J.; Van Wouwe, N.; Yang, J. H.; Aldous, K. M.

434

Perfluorooctanesulfonate and related fluorochemicals in human blood from several

435

countries. Environ. Sci. Technol. 2004, 38, 4489–4495.

436

(4)

Van de Vijver, K. I.; Hoff, P. T.; Das, K.; Van Dongen, W.; Esmans, E. L.; Jauniaux,

437

T.; Bouquegneau, J. M.; Blust, R.; de Coen, W. Perfluorinated chemicals infiltrate

438

ocean waters: link between exposure levels and stable isotope ratios in marine


Page 28 of 38

Page 29 of 38


439 440

mammals. Environ. Sci. Technol. 2003, 37, 5545–5550. (5)

Jian, J. M.; Guo, Y.; Zeng, L.; Liang-Ying, L.; Lu, X.; Wang, F.; Zeng, E. Y.; Global

441

distribution of perfluorochemicals (PFCs) in potential human exposure source-A

442

review. Environ. Int. 2017, 108, 51–62.

443

(6)

Lau, C.; Anitole, K.; Hodes, C.; Lai, D.; Pfahles-Hutchens, A.; Seed, J. Perfluoroalkyl

444

acids: a review of monitoring and toxicological findings. Toxicol. Sci., 2007, 99, 366–

445

394.

446

(7)

447 448

Lindstrom AB, Strynar MJ, Libelo EL. Polyfluorinated compounds: past, present, and future. Environ. Sci. Technol. 2011, 45, 7954–7961.

(8)

Zhang, T.; Sun, H.; Lin, Y.; Qin, X.; Zhang, Y.; Geng, X.; Kannan, K. Distribution of

449

poly- and perfluoroalkyl substances in matched samples from pregnant women and

450

carbon chain length related maternal transfer. Environ. Sci. Technol. 2013, 47, 7974–

451

7981.

452

(9)

Stockholm Convention, 2009. C.N.524.2009.TREATIES-4 (Depositary Notification).

453

(10) Wang, T.; Wang, Y.; Liao, C.; Cai, Y.; Jiang, G.; Perspectives on the inclusion of

454

perfluorooctane sulfonate into the Stockholm Convention on Persistent Organic

455

Pollutants. Environ. Sci. Technol. 2009, 43, 5171–5175.



456

(11) Peraza, M. A.; Burdick, A. D.; Marin, H. E.; Gonzalez, F. J.; Peters, J. M. The

457

toxicology of ligands for peroxisome proliferator-activated receptors (PPAR). Toxicol.

458

Sci. 2006, 90, 269–295.

459 460 461 462 463 464

(12) Mariussen E. Neurotoxic effects of perfluoroalkylated compounds: mechanisms of action and environmental relevance. Arch. Toxicol. 2012, 86, 1349–1367. (13) DeWitt JC, Peden-Adams MM, Keller JM, Germolec DR. Immunotoxicity of perfluorinated compounds: recent developments. Toxicol. Pathol. 2012, 40, 300–311. (14) Issemann, I.; Green, S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990, 347, 645–650.

465

(15) Dreyer, C.; Krey, G.; Keller, H.; Givel, F.; Helftenbein, G.; Wahli, W. Control of the

466

peroxisomal β-oxidation pathway by a novel family of nuclear hormone receptors. Cell

467

1992, 68, 879–887.

468 469

(16) Tontonoz, P.; Hu, E.; Graves, R. A.; Budavari, A. I.; Spiegelman, B. M. mPPARγ2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994, 8, 1224–1234.

470

(17) Lee, S. S.; Pineau, T.; Drago, J.; Lee, E. J.; Owens, J. W.; Kroetz, D. L.;

471

Fernandez-Salguero, P. M.; Westphal, H.; Gonzalez, F. J. Targeted disruption of the α

472

isoform of the peroxisome proliferators-activated receptor gene in mice results in


Page 30 of 38

Page 31 of 38


473

abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell Biol. 1995,

474

15, 3012–3022.

475

(18) Waxman, D. J. P450 gene induction by structurally diverse xenochemicals: central role

476

of nuclear receptors CAR, PXR, and PPAR. Arch. Biochem. Biophys. 1999, 369, 11–

477

23.

478

(19) Diaz, M. J.; Chinje, E.; Kentish, P.; Jarnot, B.; George, M.; Gibson, G. Induction of

479

cytochrome P4504A by the peroxisome proliferators perfluoro-n-octanoic acid.

480

Toxicology 1994, 86, 109–122.

481

(20) Buhrke, T.; Kibellus, A.; Lampen, A. In vitro toxicological characterization of

482

perfluorinated carboxylic acids with different carbon chain lengths. Toxicol. Lett. 2013,

483

218, 97–104.

484

(21) Vanden Heuvel, J. P.; Thompson, J. T.; Frame, S. R.; Gillies, P. J. Differential

485

activation of nuclear receptors by perfluorinated fatty acid analogs and natural fatty

486

acids: a comparison of human, mouse, and rat peroxisome proliferators-activated

487

receptor-α, -β, and -γ, liver X receptor-β, and retinoid X receptor-α. Toxicol. Sci. 2006,

488

92, 476–489.

489

(22) Wolf, C. J.; Takacs, M. L.; Schmid, J. E.; Lau, C.; Abbott, B. D. Activation of mouse



490

and human peroxisome proliferator-activated receptor alpha by perfluoroalkyl acids of

491

different functional groups and chain lengths. Toxicol. Sci., 2008, 106, 162–171.

492

(23) Wolf, C. J.; Schmid, J. E.; Lau, C.; Abbott, B. D. Activation of mouse and human

493

peroxisome proliferator-activated receptor-alpha (PPARα) by perfluoroalkyl acids

494

(PFAAs): further investigation of C4-C12 compounds. Reprod. Toxicol. 2012, 33, 546–

495

551.

496

(24) Iwata, H.; Watanabe, M.; Okajima, Y.; Tanabe, S.; Amano, M.; Miyazaki, N.; Petrov,

497

E. A. Toxicokinetics of PCDD, PCDF, and coplanar PCB congeners in Baikal seals,

498

Pusa sibirica: age-related accumulation, maternal transfer, and hepatic sequestration.

499

Environ. Sci. Technol. 2004, 38, 3505–3513.

500

(25) Imaeda, D.; Kunisue, T.; Ochi, Y.; Iwata, H.; Tsydenova, O.; Takahashi, S.; Amano,

501

M.; Petrov, E. A.; Batoev, V. B.; Tanabe, S. Accumulation features and temporal trends

502

of PCDDs, PCDFs and PCBs in Baikal seals (Pusa sibirica). Environ. Pollut. 2009,

503

157, 737–747.

504

(26) Nakata, H.; Tanabe, S.; Tatsukawa, R.; Amano, M.; Miyazaki, N.; Petrov, E. A.

505

Persistent organochlorine residues and their accumulation kinetics in Baikal seal

506

(Phoca sibirica) from Lake Baikal, Russia. Environ. Sci. Technol. 1995, 29, 2877–


Page 32 of 38

Page 33 of 38

507


2885.

508

(27) Ishibashi, H.; Iwata, H.; Kim, E. Y.; Tao, L.; Kannan, K.; Amano, M.; Miyazaki, N.;

509

Tanabe, S.; Batoev, V. B.; Petrov, E. A. Contamination and effects of

510

perfluorochemicals in Baikal seal (Pusa sibirica). 1. residue level, tissue distribution,

511

and temporal trend. Environ. Sci. Technol. 2008, 42, 2295–2301.

512

(28) Ishibashi, H.; Iwata, H.; Kim, E. Y.; Tao, L.; Kannan, K.; Tanabe, S.; Batoev, V. B.;

513

Petrov, E. A. Contamination and effects of perfluorochemicals in Baikal seal (Pusa

514

sibirica). 2. molecular characterization, expression level, and transcriptional activation

515

of peroxisome proliferator-activated receptor α. Environ. Sci. Technol. 2008, 42, 2302–

516

2308.

517

(29) Ishibashi, H.; Kim, E. Y.; Iwata, H. Transactivation potencies of the Baikal seal (Pusa

518

sibirica) peroxisome proliferator-activated receptor α by perfluoroalkyl carboxylates

519

and sulfonates: estimation of PFOA induction equivalency factors. Environ. Sci.

520

Technol. 2011, 45, 3123–3130.

521

(30) Bernardes, A.; Souza, P. C.; Muniz, J. R.; Ricci, C. G.; Ayers, S. D.; Parekh, N. M.;

522

Godoy, A. S.; Trivella, D. B.; Reinach, P.; Webb, P.; Skaf, M. S.; Polikarpov, I.

523

Molecular mechanism of peroxisome proliferator-activated receptor α activation by



524

WY14643: a new mode of ligand recognition and receptor stabilization. J. Mol. Biol.

525

2013, 425 (16), 2878–2893.

526

(31) Faiola, B.; Falls, J. G.; Peterson, R. A.; Bordelon, N. R.; Brodie, T. A.; Cummings, C.

527

A.; Romach, E. H.; Miller, R. T. PPAR alpha, more than PPAR delta, mediates the

528

hepatic and skeletal muscle alterations induced by the PPAR agonist GW0742. Toxicol.

529

Sci. 2008, 105 (2), 384–394.

530

(32) Sznaidman, M. L.; Haffner, C. D.; Maloney, P. R.; Fivush, A.; Chao, E.; Goreham, D.;

531

Sierra, M. L.; LeGrumelec, C.; Xu, H. E.; Montana, V. G.; Lambert, M. H.; Willson, T.

532

M.; Oliver, W. R. Jr.; Sternbach, D. D. Novel selective small molecule agonists for

533

peroxisome proliferator-activated receptor delta (PPARdelta)--synthesis and biological

534

activity. Bioorg. Med. Chem. Lett. 2003, 13 (9), 1517–1521.

535 536

(33) Endo, Y.; Sawasaki, T. Advances in genome-wide protein expression using the wheat germ cell-free system. Methods Mol. Biol. 2005, 310, 145–167.

537

(34) Dau, P. T., Sakai, H., Hirano, M., Ishibashi, H., Tanaka, Y., Kameda, K., Fujino, T.,

538

Kim, E. Y., Iwata, H. Quantitative analysis of the interaction of constitutive androstane

539

receptor with chemicals and steroid receptor coactivator 1 using surface plasmon

540

resonance biosensor systems: a case study of the Baikal seal (Pusa sibirica) and the


Page 34 of 38

Page 35 of 38

541


mouse. Toxicol. Sci. 2013, 131 (1), 116–127.

542

(35) Hirano, M., Hwang, J. H., Park, H. J., Bak, S. M., Iwata, H., Kim, E. Y. In silico

543

analysis of the interaction of avian aryl hydrocarbon receptors and dioxins to decipher

544

isoform-, ligand-, and species-specific activations. Environ. Sci. Technol. 2015, 49 (6),

545

3795–3804.

546

(36) Hostetler, H. A.; Petrescu, A. D.; Kier, A. B.; Schroeder, F. Peroxisome

547

proliferator-activated receptor α interacts with high affinity and is conformationally

548

responsive to endogenous ligands. J. Biol. Chem. 2005, 280, 18667–18682.

549

(37) Velkov, T.; Rimmer, K. A.; Headey, S. J. Ligand-enhanced expression and in-cell assay

550

of human peroxisome proliferator-activated receptor alpha ligand binding domain.

551

Protein Expr. Purif. 2010, 70, 260–269.

552 553 554 555 556 557

(38) Endo, Y.; Sawasaki, T. Cell-free expression systems for eukaryotic protein production. Curr. Opin. Biotechnol. 2006, 17 (4), 373–380. (39) https://tools.thermofisher.com/content/sfs/manuals/lanthascreen_PPARalpha_competiti vebinding_man.pdf. (40) Li, C. H.; Ren, X. M.; Ruan, T.; Cao, L. Y.; Xin, Y.; Guo, L. H.; Jiang, G. Chlorinated polyfluorinated

ether

sulfonates

exhibit

higher


activity

toward

peroxisome


558

proliferator-activated receptors signaling pathways than perfluorooctanesulfonate.

559

Environ. Sci. Technol. 2018, 52 (5), 3232–3239.

560

(41) Luebker, D. J.; Hansen, K. J.; Bass, N. M.; Butenhoff, J. L.; Seacat, A. M. Interactions

561

of fluorochemicals with rat liver fatty acid-binding protein. Toxicology 2002, 176, 175–

562

185.

563

(42) Jones, P. D.; Hu, W.; De Coen, W.; Newsted, J. L.; Giesy, J. P. Binding of

564

perfluorinated fatty acids to serum proteins. Environ. Toxicol. Chem. 2003, 22, 2639–

565

2649.

566

(43) Beesoon, S.; Martin, J. W. Isomer-specific binding affinity of perfluorooctanesulfonate

567

(PFOS) and perfluorooctanoate (PFOA) to serum proteins. Environ. Sci. Technol. 2015,

568

49, 5722–5731.

569

(44) Chen, H.; He, P.; Rao, H.; Wang, F.; Liu, H.; Yao, J. Systematic investigation of the

570

toxic mechanism of PFOA and PFOS on bovine serum albumin by spectroscopic and

571

molecular modeling. Chemosphere 2015, 129, 217–224.

572 573 574

(45) Han, X.; Snow, T. A.; Kemper, R. A.; Jepson, G. W. Binding of perfluorooctanoic acid to rat and human plasma proteins. Chem. Res. Toxicol. 2003, 16 (6), 775–781. (46) Zhang, L.; Ren, X. M.; Guo, L. H. Structure-based investigation on the interaction of


Page 36 of 38

Page 37 of 38


575

perfluorinated compounds with human liver fatty acid binding protein. Environ. Sci

576

Technol. 2013, 47 (19), 11293–11301.

577 578

(47) Chen, Y. M.; Guo, L. H. Fluorescence study on site-specific binding of perfluoroalkyl acids to human serum albumin. Arch. Toxicol. 2009, 83 (3), 255–261.

579

(48) Bischel, H. N.; Macmanus-Spencer, L. A.; Luthy, R. G. Noncovalent interactions of

580

long-chain perfluoroalkyl acids with serum albumin. Environ. Sci. Technol. 2010, 44

581

(13), 5263–5269.

582

(49) Xu, H. E.; Lambert, M. H.; Montana, V. G.; Parks, D. J.; Blanchard, S. G.; Brown, P.

583

J.; Sternbach, D. D.; Lehmann, J. M.; Wisely, G. B.; Willson, T. M.; Kliewer, S. A.;

584

Milburn,

585

proliferator-activated receptors. Mol. Cell. 1999, 3, 397–403.

M.

V.

Molecular

recognition

of

fatty

acids

by

peroxisome

586

(50) Xu, H. E.; Lambert, M. H.; Montana, V. G.; Plunket, K. D.; Moore, L. B.; Collins, J.

587

L.; Oplinger, J. A.; Kliewer, S. A.; Gampe, R. T.; McKee Jr, D. D.; Moore, J. T.;

588

Willson, T. M. Structural determinants of ligand binding selectivity between the

589

peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. U. S. A. 2001, 98

590

(24), 13919–13924.



591 592

Table of Contents (TOC) Art

593


Page 38 of 38

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl

Recommend Documents