Endocannabinoid Interaction with Human FABP1: Impact of the T94A

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Endocannabinoid Interaction with Human FABP1: Impact of T94A Variant Gregory Gerald Martin, Huan Huang, Avery Lee McIntosh, Ann B. Kier, and Friedhelm Schroeder Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00647 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on September 4, 2017

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Biochemistry

Endocannabinoid Interaction with Human FABP1: Impact of T94A Variant

by Gregory G. Martin*, Huan Huang*, Avery L. McIntosh*, Ann B. Kier‡, and Friedhelm Schroeder*,1

from the *

Department of Physiology and Pharmacology,

Texas A&M University, College Station, TX 77843-4466 and the ‡

Department of Pathobiology,

Texas A&M University, College Station, TX 77843-4467

Running Title: FABP1 T94A variant alters endocannabinoid interactions

1

Address Correspondence to:

Friedhelm Schroeder, Department of Physiology and

Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466. Phone: (979) 862-1433, FAX: (979) 862-4929; E-mail: [email protected]

Keywords: mouse, liver, FABP1, endocannabinoid, binding, structure

1 ACS Paragon Plus Environment

Biochemistry

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Abbreviations:

AEA,

n-6

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arachidonoylethanolamide

(anandamide);

2-AG,

2-

arachidonoylglycerol; ANS, 1-anilinonaphthalene-8-sulfonic acid; CB1, cannabinoid receptor-1; cis-PnCoA, cis-parinaroyl-CoA; DAUDA, 11-(dansylamino)-undecanoic acid; DHEA, n-3 docosahexaenoylethanolamide; EC, endocannabinoid; EPEA, n-3 eicosapentaenoylethanolamide; FAAH, fatty acid amide hydrolase; FABP1 T94T, human liver fatty acid binding protein-1 T94T (wild-type FABP1 T94T; also called L-FABP T94T); FABP1 T94A, human liver fatty acid binding protein-1 T94A (FABP1 T94A variant); FABP3, fatty acid binding protein-3; FABP5, fatty acid binding protein5; FABP7, fatty acid binding protein-7; LCFA, long chain fatty acid; LCFA-CoA, long chain fatty acyl CoA; 2-MG, 2-monoacylglycerol; MGL, 2-monoacylglycerol lipase; NAE, N-acylethanolamides; NAFLD, non-alcohol fatty liver disease; NBD-stearic acid, [20-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]

stearic

arachidonoylethanolamide

[20-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]

or

acid;

NBD-AEA,

NBD-N-

arachidonoylethanolamide; NBD-2-AG, NBD-2-arachidonoylglycerol or 2-[20-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino]

arachidonoylglycerol;

OEA,

oleoylethanolamide;

2-OG,

2-

oleoylglycerol; PEA, palmitoylethanolamide; 2-PG, 2-palmitoylglycerol; PPARα peroxisome proliferator-activated receptor alpha; SCP-2, sterol carrier protein-2; SCP-x, sterol carrier proteinx.


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Biochemistry

ABSTRACT Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide (i.e. NBD-AEA) and 2arachidonoylglycerol (NBD-2-AG) with high affinity. Most native N-acylethanolamides (NAEs), but only one 2-monoacylglycerol (i.e. 2-arachidonoylglycerol, 2-AG) displaced WT FABP1-bound fluorescently labeled endocannabinoids (ECs). While T94A variant did not differ in affinity for AEA and most other NAE, it exhibited modestly higher affinity for OEA, as well as higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1’s secondary structure more extensively than any other previous ligand examined. The T94A variant without ligand was more susceptible to temperatureinduced unfolding. While the T94A variant was much less sensitive to ligand (i.e. AEA, 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized T94A structure to thermal unfolding. These data provide the first evidence that ECs not only bind to but also alter the secondary structure of the human FABP1—with the latter markedly impacted by the T94A substitution, a variant highly associated with hepatic accumulation of lipids and non-alcoholic fatty liver disease (NAFLD). Importantly, NAFLD has been associated with elevated hepatic levels of ECs and FABP1.


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INTRODUCTION Almost all previous reports characterizing ligand interactions and structure of human fatty acid binding protein-1 (FABP1) were performed with the native FABP1 or with recombinant proteins produced via cDNAs encoding the WT T94T FABP1 2-4. Consequently, findings on human FABP1 ligand specificity

5-7

, structure

8-11

, and mode of ligand binding

7,10,11

reflected those of recombinant human WT

T94T FABP1 protein. These studies demonstrated that the human FABP1 is unique, not only as compared to other species FABP1s, but also among known members of the FABP family in having the largest binding cavity heretofore discovered for any FABP

7-11

. Importantly, human FABP1’s conformational

flexibility and mode of long chain fatty acid (LCFA) binding likewise differ significantly from that of other species’ FABP1s 8-11. A SNP in the human Fabp1 gene coding sequence leading to a human FABP T94A substitution is the most prevalent polymorphism in the FABP family (26-38% minor allele freq.; 8.3±1.9% homozygous; MAF for 1000 genomes in NCBI dbSNP database; ALFRED database) acid substitution has little overall effect on overall tertiary structure

12-18

19-21

. This T94A single amino or, with the exception of

cholesterol, the affinity for most lipidic ligands (LCFA, LCFA-CoA, lysophospholipids, fibrates)

19,20,22

.

On the other hand, the T94A substitution significantly alters distal amino acid orientations/interactions and secondary structures differentially induced by binding of such ligands

19,20,22

Expression of the

T94A variant is associated with neutral lipid accumulation (e.g. triacylglycerides) in transfected Chang liver cells

23

and cultured primary human hepatocytes

24

as well as non-alcoholic fatty liver disease

(NAFLD) in human subjects 17. The molecular link(s) between the human FABP1 and NAFLD are only beginning to be understood. Several studies suggest involvement of the endocannabinoid (EC) system as a possibility. Hepatic levels of the EC receptor CB1 25-27, ECs such as AEA 25,26, and FABP1 28-31 are elevated in human NAFLD and in animal models of NAFLD. In fact, AEA activation of CB1 receptors is thought to be required for development of NAFLD in mice

25,26,32

. However, until recently it was unclear as to how the very

hydrophobic ECs such as AEA trafficked within the cell for targeting to CB1 at the plasma membrane or 4 ACS Paragon Plus Environment

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Biochemistry

other proteins within other cellular organelles (e.g. peroxisome proliferator activated receptor-α, PPARα). A novel discovery demonstrated that rat FABP1 bound EC (e.g. AEA, 2-AG) with high affinity 1

. Moreover, Fabp1 gene ablation markedly altered hepatic EC levels in mice 1. While this indicated that

FABP1 is the major EC binding/‘chaperone’ protein in murine liver, the markedly different ligand binding cavity of the human liver FABP1 8-11 suggests that it may not be appropriate to simply extend rat FABP1’s EC binding affinity/specificity to that of the human FABP1, much less to the human T94A variant. Ligand (e.g. LCFA, LCFA-CoA, fibrates) induced conformational changes in the murine FABP1 are essential for its functional interactions with other intracellular proteins. Studies with recombinant murine FABP1 and cultured cells and primary hepatocytes indicate that binding of such ligands alters murine FABP1’s conformation to facilitate direct interaction with target proteins involved in hepatic lipid metabolism including: fatty acid translocase protein-5 at the plasma membrane

33

, carnitine palmitoyl

acyltransferase-1A at the outer mitochondrial membrane (the rate limiting enzyme in mitochondrial LCFA oxidation) 34, and peroxisome proliferator activated receptor-α (PPARα) in the nucleus where it activates transcription of multiple enzymes in fatty acid oxidation and lipid metabolism 35-39. Recent NMR studies established for the first time that the fibrate binding also altered the conformation of human WT FABP1 to facilitate interaction with PPARα for fibrate transfer and induction of PPARα transcriptional activity 7. The T94A substitution in human FABP1 markedly diminishes the ability of ligands such as LCFA, LCFA-CoA, and fibrate to alter FABP1 conformation and activate PPARα 19,20,20. Again, nothing is known regarding the ability of ECs to alter the conformation of human WT FABP1, much less the T94A variant. Based on the remarkable differences between murine and human FABP1 structure and ligand specificity, the study presented herein was undertaken to determine: i) the extent to which human WT FABP1 bound both NAEs and 2-MGs; ii) ability of NAEs and 2-MGs to alter human WT FABP1


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secondary structure; iii) impact of the T94A substitution on EC-induced alterations in human FABP1 secondary structure and stability.

MATERIALS AND METHODS Materials.

n-6

palmitoylethanolamide

arachidonoylethanolamide (PEA),

n-3

(AEA),

oleoylethanolamide

docosahexaenoylethanolamide

(OEA),

(DHEA),

n-3

eicosapentaenoylethanolamide (EPEA), 2-arachidonoylglycerol (2-AG), 2-oleoylglycerol (2-OG), and 2palmitoylglycerol (2-PG) were acquired from Cayman Chemical (Ann Arbor, MI). The fluorescent probe 11-(dansylamino) undecanoic acid (DAUDA) was purchased from Cayman Chemical (Ann Arbor, MI). Cis-parinaric acid was acquired from Invitrogen/Life Technologies (Grand Island, NY, USA) and used to synthesize cis-parinaroyl-CoA40. ANS (1-anilinonaphthalene-8-sulfonic acid) was obtained from Life Technologies (Grand Island, NY). NBD-AEA (NBD-N-arachidonoylethanolamide or [20-[(7-nitro-2-1,3benzoxadiazol-4-yl)amino] arachidonoylethanolamide) and NBD-2-AG (NBD-2-arachidonoylglycerol or 2-[20-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]

arachidonoylglycerol

were

synthesized

by

Dr.

Shengrong Li and generously provided by Drs. Stephen Burgess and Walt Shaw (Avanti Polar Lipids, Alabaster, AL). NBD-stearic acid [12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid] was also from Avanti Polar Lipids (Alabaster, AL). All solvents and reagents used were of the highest commercial grade available. Recombinant human FABP1 T94T and FABP1 T94A proteins. The cDNA encoding human FABP1 was obtained from OriGene (Rockville, MD). The commercially available cDNA was shown to code for the human FABP1 T94A mutant and was used to prepare the recombinant human FABP1 T94T (WT) using standard mutagenesis procedures24. Recombinant human FABP1 for both was prepared from these cDNA24. Purity and identity of the recombinant human FABP1 T94T and FABP1 T94A proteins was established by sequencing (Gene Tech. Lab, Texas A&M University) and MS/proteomics (LBMS Lab, Texas A&M University) 24. NAE and 2-MG binding to recombinant human FABP1 T94T and FABP1 T94A proteins. 6 ACS Paragon Plus Environment

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Biochemistry

Since native ECs (AEA, 2-AG) are known to displace several fluorescent ligands bound to WT rat FABP1 including: cis-parinaroyl-CoA1,41,42, ANS20, and DAUDA1,43, these ligands were used in displacement assays to examine WT human FABP1 T94T or FABP1 T94A variant protein binding characteristics. In addition, NBD-AEA and NBD-2-AG probes were developed to determine direct binding affinities to WT human FABP1 T94T or FABP1 T94A analogous to that for NBD-stearic acid binding to WT rat FABP1 44. For reverse titration, the respective NBD-labeled probes (100nM) were titrated with increasing WT or T94A variant FABP1 (0-3µM). For forward titration, WT or T94A variant FABP1 (250nM) was titrated with NBD-labeled probe (0-1.5 µM). The NBD probes in 10mM potassium phosphate buffer (pH=7.4) with or without human FABP1 were excited at 490nm while emission was scanned from 515600nm. Samples in quartz cuvettes (VWR, Radnor, PA) were maintained at 24oC using a Peltier controlled cell holder and spectra were obtained using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Data were corrected for blanks/controls including: fluorescent ligand only, FABP1 T94T (or FABP1 T94A) protein only, and photobleaching. The saturation maximum of the reverse titration curve was calculated using curve fitting in order to obtain the fluorescence intensity (per nM) of each NBD-labeled probe when fully bound to WT or T94A variant FABP1. This important parameter, when used together with the forward titration, allows determination of the fractional saturation and free NBD-labeled probe concentration. Converting the forward titration data thus allows plotting the forward titration as fractional saturation (vertical axis) vs free NBD-probe concentration (horizontal axis). The curves can now be fit to calculate the binding parameters: Kd and Bmax. NBD-AEA and NBD-2-AG binding curves to WT and T94A variant FABP1 were each best fit by a single component equation analogous to WT rat FABP1 binding NBD-cholesterol 45. In the corresponding displacement assays, human FABP1 (250nM) was equilibrated with NBD-AEA or NBD-2-AG (1 µM), then the protein - fluorescent ligand complex was titrated with NAE or 2-MG. Fluorescence intensity of NBD-AEA and NBD-2-AG was recorded after each addition of displacing ligand as described above. Each data point was corrected by subtracting the signals of protein in buffer 7 ACS Paragon Plus Environment

Biochemistry

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only, and signals of NBD-AEA and NBD-2-AG plus increasing amount of displacing ligand in the absence of the proteins. Displacement curves were constructed by plotting the percentage of NBD-AEA or NBD-2-AG fluorescence remaining (Y) after all the corrections was made (at emission maximum 540nm) versus ligand concentration (X). The displacement binding dissociation constant Ki is determined according to the equation: Ki,ligand = (Kd,NBD x EC50)/[NBD]total, where Kd,NBD is the binding affinity (dissociation constant) of NBD-AEA or NBD-2-AG to human FABP1 (obtained above), EC50 is the ligand concentration at which half of the maximum NBD-AEA or NBA-2-AG displacement occurred, [NBD]total (1µM) is the total NBD-AEA or NBD-2-AG concentration used in the displacement assays. Impact of NAE and 2-MG binding on recombinant human WT FABP1 T94T and FABP1 T94A variant protein secondary structure: circular dichroism (CD). The impact of NAE and 2-MG binding on human FABP1 T94T and FABP1 T94A variant secondary structure was determined by circular dichroism similarly as described earlier for the binding of other ligands (fatty acids, fatty acylCoAs, fibrates) to rat FABP1

19

. Briefly, CD spectroscopy was performed with a JASCO J-815 CD

spectrometer (JASCO, Easton, MD) equipped with a Model PFD-425S Peltier Type FDCD attachment for temperature regulation. FABP1 T94T (or FABP1 T94A) protein concentrations were performed by amino acid analysis (Dr. Larry Dangott, Protein Chemistry Laboratory, Texas A&M University, College Station, TX). All NAE and 2-MG binding experiments were performed at 0.5 µM WT FABP1 T94T (or FABP1 T94A variant) protein in 10 mM potassium phosphate (pH 7.4) by titrating the each protein with increasing amount of NAE or 2-MG from stock solutions of 500 µM ligand in ethanol such that ligand and ethanol final concentrations were 5 µM and 1%, respectively. Each protein/ligand sample was placed in the FDCD attachment and incubated at 25 oC with stirring for 10 min before the CD spectrum was obtained.

Each final CD spectrum represented the average of ten scans that were background

(buffer/ligand/ethanol) subtracted, mathematically smoothed, and secondary structure determined as described 19. The % change in secondary structure upon ligand binding was calculated using the following formula: [(% Secondary StructureUnliganded - % Secondary StructureLigand) ÷ % Secondary


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Biochemistry

StructureUnliganded] * 100%. The presence of ethanol was determined to have no effect on ligand binding, protein CD spectra, or resulting secondary structure determinations (not shown). Effect of AEA and 2-AG binding on thermal stability of and change in secondary structure of recombinant human WT FABP1 T94T and FABP1 T94A variant protein: circular dichroism (CD) with increasing temperature. Circular dichroism temperature studies of human WT FABP1 and FABP1 T94A variant in the presence and absence of NAE or 2-MG were performed as follows: Human WT FABP1 and FABP1 T94A variant protein (0.5 µM protein in 10 mM potassium phosphate, pH 7.4) was incubated without or with 5 µM AEA or 5 µM 2-AG with stirring at 25 oC for 10 min in the FDCD attachment for obtaining CD spectra as described in the preceding section. Sample temperature was increased by 10 oC at a rate of 1 oC/min followed by an additional 10 min incubation at each temperature prior to obtaining the CD spectrum. This procedure was repeated until the final sample at 95 oC. CD spectra were then analyzed and secondary structure determinations were performed as described in the preceding section. Statistical Analysis. All values represent the mean ± SEM. Statistical analysis was performed by simple Student’s t-test. Differences of P < 0.05 were considered statistically significant.

RESULTS Binding of NAEs to FABP1: displacement of protein-bound fluorescent ligands by nonfluorescent ligands. The ligand binding cavity of the human WT FABP1 is the largest of any known FABP family member

7-11,46-48

—suggesting it may differ significantly from rat FABP1 in affinity for

NAEs. This possibility was examined with human WT FABP1 T94T analogous to that described for displacement of the following fluorophores bound to rat WT FABP1: cis-parinaroyl-CoA 1,41,42; ANS 20; NBD-stearic acid 44; DAUDA 1,43.


Biochemistry

Neither AEA nor OEA significantly displaced bound cis-parinaroyl-CoA from human WT FABP1 (not shown)—in marked contrast to displacement of cis-parinaroyl-CoA from rat WT FABP1

1,41,42

.

Likewise, AEA and OEA did not displace NBD-stearic acid bound to human WT FABP1 (not shown)— again in contrast to displacement of NBD-stearic acid from other members of the FABP family (i.e. FABP3,5, and 7)

49,50

. In addition, AEA and OEA also did not displace ANS bound to rat WT FABP1

(not shown)—consistent with the inability of such ligands to displace ANS bound to rat WT FABP1 1. Finally, both AEA and OEA only partially (about 30%) displaced DAUDA bound to human WT FABP1—consistent with only partial (13%) displacement of DAUDA bound to rat WT FABP11.

40

Fluorescence Intensity (A.U.)

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NBD-AEA

A

+ T94A

30 + WT

20 10

In buffer

0

B

NBD-2-AG

30

+ T94A

20

+ WT

10 In buffer

0 520

540

560

580

600

Wavelength (nm) Figure 1. Spectral properties of NBD-labeled ECs bound to human WT FABP1 T94T and FABP1 T94A variant. Representative fluorescence emission spectra (515-600nm with 490 nm excitation) of NBD-AEA (Panel A) and NBD-2-AG (Panel B) in the absence (solid lines) or presence of human WT FABP1 (dotted lines) and FABP1 T94A variant (dashed lines) were determined as described in Methods. The concentrations for NBD-AEA and NBD-2-AG were 100nM. The protein concentrations were 2.7 µM for incubation with NBD-AEA, and 2µM for incubation with NBD-2-AG. Representative spectra are shown. Taken together, these displacement data indicated either the human WT FABP1 did not bind NAE or that the NAE binding site of the human WT FABP1 differed markedly from that of the rat WT FABP1. 10 ACS Paragon Plus Environment

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Biochemistry

This issue was addressed by the development of NBD-labeled EC analogues for direct binding assays as well as displacement assays by native NAE and 2-MG. Effect of fluorescent NBD-AEA binding to human FABP1 and T94A variant: Blue-shifted maximal emission wavelengths. The possibility that human WT FABP1 and T94A variant might bind a recently-developed novel NBD-labeled analogue of AEA, i.e. NBD-AEA

1,51

, was determined by

examining the fluorescence emission spectral properties of this probe in buffer alone and in the presence of FABP1 as described in Methods.

Table 1. Human FABP1 binding impact on NBD-labeled ECs spectral properties: ‘blue’-shifted emission maxima. Fluorescence emission spectra of NBD-AEA (100nM) and NBD-2-AG (100nM) were obtained in the absence or presence of human WT FABP1 or T94A variant protein as described in Methods. Protein concentrations were 2.7µM for incubation with NBD-AEA and 2µM for incubation with NBD-2-AG. Maximal fluorescence emission wavelength ‘blue-shift’ were determined for each spectrum as shown in parenthesis. Values are the mean ± SEM, n=3-7. * refers to p> PEA) was the same as for the WT FABP1.

Table 3. Affinity of human WT FABP1 and T94A variant proteins for NAEs and 2-MGs: Displacement of bound NBD-AEA and NBD-2-AG: Kis were determined from displacement of FABP1-bound fluorescent NBD-AEA for the NAEs and for displacement of FABP1-bound fluorescent NBD-2-AG for 2-MGs as described in Methods. ND = no significant displacement. Values are the mean ± SEM, n=4. * refers to p> PEA. T94A substitution significantly altered the impact of NAE binding on FABP1 secondary structure in a NAE species-specific manner in the overall order: AEA> DHEA, EPEA > OEA > PEA. Binding of 2-AG selectively altered the secondary structure of human WT FABP1 T94T protein: Impact of the T94A substitution. Studies with murine FABP1 have shown that binding of 2MG also alters the secondary structure of this protein 1. Therefore, the effect (if any) of 2-MG binding on human WT FABP1’s secondary structure or the impact of T94A thereon was examined with 2-AG (n-6), 2-OG (n-9), and 2-PG (saturated). Binding of 2-AG to human WT FABP1 markedly increased the proportion of distorted α-helix by 45% (Figure 8A), decreased total β-sheet by 10% (primarily regular β-sheet) (Figure 8B), and turns by 15% (Figure 8C). T94A substitution markedly increased the impact of 2-AG binding on proportion of total α-helix, regular α-helix, and especially distorted α-helix (Figure 8A) as well as unordered structure (Figure 8C). Binding of 2-AG to FABP1 T94A resulted in a modest increase in regular β-sheet (Figure 8B). The binding of 2-OG (Figure 8D-F) or 2-PG (Figure 8G-I) had very little impact on the secondary structure of human WT FABP1 or by the T94A substitution—consistent with their weak/no binding. Binding of 2-MGs to human WT FABP1 and T94A variant provided several new insights: i) 2-AG altered the secondary structure of the human WT FABP1 more so than any other EC or EC analogue tested, regardless of whether NAE or 2-MG; ii) T94A substitution much more dramatically altered secondary structure as compared to others tested.


Biochemistry

200

Ligand Induced Change in Secondary Structure (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2-AG A T94T T94A

150

*

100

*

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2-PG

2-OG α-Helix

D

G

*

50

*

0 -50

B

*

E

β -Sheet

H

*

*

*

*

40 20

*

*

*

0 -20 50

Regular Distorted Total


C

F

*

0 -50

*


I

*

*

*

-100

*

-150 Turns

Unordered

Turns

Unordered

Turns

Unordered

Figure 8. Changes in human FABP1 WT and FABP1 T94A variant secondary structure induced by binding 2-MG. CD of recombinant human FABP1 T94T or FABP1 T94A variant (0.5 µM) protein in the absence or presence of 5 µM ligand as described in Methods. Results were expressed as % change in secondary structure (FABP1/ligand – FABP1 only) of human WT FABP1 T94T (solid black bars), and FABP1 T94A variant (open bars) upon interaction with 2-AG (Panels A-C), 2-OG (Panels DF), and 2-PG (Panels G-I). Panels A,D, and G show the % change in regular, distorted and total α-helix. Panels B,E, and H show the % change in regular, distorted and total β-sheet. Panels C, F, and I show the % change in turns and unordered structures. *, P < 0.05 for FABP1 T94A/ligand secondary structure versus FABP1 T94T/ligand secondary structure. Effect of AEA and 2-AG binding on thermal stability of human WT FABP1 T94T and FABP1 T94A variant protein secondary structure. It is thought that ligand-induced conformational changes in murine and/or human WT FABP1 facilitate FABP1 interaction with and ligand transfer to proteins such as carnitine palmitoyl acyltransferase 1A to facilitate fatty acid oxidation transcription of fatty acid oxidative genes

7,35-39,52

34,34

or with PPARα to induce

. However, it is not known if the NAE and 2-MG

induced changes in human WT and/or T94A variant proteins not only confer a favorable conformation but also stabilize their secondary structure to promote functional interaction. Therefore, the ability of ECs to stabilize the secondary structure of human WT and T94A variant was determined by examining the response of the proteins (with and without ligand) to thermal-induced unfolding. 22 ACS Paragon Plus Environment

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Biochemistry

In the absence of ligand, increasing temperature decreased the human WT FABP1 proportion of αhelix (Figure 9A), less so β-sheet (Figure 9D), and relatively little turns (Figure 9G)—all at the expense of dramatically increasing unordered structure (Figure 9J). The T94A substitution significantly decreased human FABP1’s proportion of α-helix (Figure 9A) and β-sheet (Figure 9D) at nearly all temperatures and induced a prominent break near 55-60°C barely or not detectable in the WT FABP1 (Figure 9A). Concomitantly, the proportion of unordered structure of the FABP1 T94A variant was greater at all temperatures as compared to the WT FABP1 and also exhibited a prominent break near 55-60°C not detectable in the WT FABP1 (Figure 9A). The T94A substitution had much less if any effect on the proportion of turn structure at most temperatures examined (Figure 9G). In the presence of bound AEA, increasing temperature also decreased the human WT FABP1 secondary structure proportion of α-helix (Figure 9B), less so β-sheet (Figure 9E), and relatively little turns (Figure 9H)—again all at the expense of dramatically increasing unordered structure (Figure 9K). Moreover, the AEA bound to WT FABP1 shifted to or induced a break in the temperature curve for the proportion of α-helix (Figure 9B) and β-sheet (Figure 9E) to higher temperature near 80-85°C, while inducing a break in unordered structure in the temperature curve near 45-50 °C (Figure 9K). AEA binding to T94A variant lowered the break in the temperature curve of α-helix (Figure 9B) and β-sheet (Figure 9D) to near 60-70°C, and even lowered that for turns (Figure 9H) and unordered structure (Figure 9K) to near 35-40 °C. In the presence of bound 2-AG, increasing temperature again decreased the human WT FABP1 secondary structure proportion of α-helix (Figure 9C), less so β-sheet (Figure 9F), and relatively little turns (Figure 9I)—also at the expense of dramatically increasing unordered structure (Figure 9L). Moreover, the bound 2-AG shifted to or induced a break in the α-helix (Figure 9C) and β-sheet (Figure 9F) temperature curves at higher temperature near 65-80 oC, while inducing a break in unordered structure in the temperature curve near 35-45 oC (Figure 9K).


Biochemistry

α-Helix (% of Total)

60

β -Sheet (% of Total)

No Ligand

B

*

T94T T94A

C

AEA

2-AG

* *

*

*

20

* * *

* * *

* E

D

F

* * *

40

*

* *

20 0

Turns (% of Total)

A

40

0

*

*

G

*

*

* * *

* I

H

40 20 0

Unordered (% of Total)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

*

J

* *

60

* * * K

L

*

40

*

* * * 20 * *

0 20

40

60

80

20

*

*

40

60

80

20

Temperature, oC

40

60

80 100

FIGURE 9. Effect of AEA and 2-AG binding on thermal stability of human WT FABP1 and T94A variant. All conditions were as described in legend to Fig. 6 except that CD measurements of recombinant human FABP1 T94T or FABP1 T94A variant (0.5 µM) protein were made every 50C over the range of 24-95 5°C in the absence or presence of 5 µM AEA or 2-AG as described in Methods. Results at each temperature point were expressed as % of total secondary structure represented by αhelix, β-sheet, turns, and unordered structure of human WT FABP1 T94T (solid black bars), and FABP1 T94A variant (open bars) in without ligand (Panels A,D,G,J), with AEA (Panels B,E,H,K), or with 2-AG (Panels C,F,I,L). Values represent the mean ± SEM, n=6. *, P < 0.05 for FABP1 T94A vs WT FABP T94T. Effect of AEA and 2-AG ligand binding on % change in secondary structure of human WT FABP1 T94T and human FABP1 T94A variant protein with increasing temperature. To more dramatically illustrate the impact of AEA and 2-AG binding on temperature sensitivity, the data were plotted for each ligand-induced change (%) in proportion of α-helix, β-sheet, turns, and unordered structure, respectively, for WT FABP1 and FABP1 T94A variant as a function of increased temperature.


Page 25 of 38

AEA binding to FABP1 T94A variant much more markedly altered its secondary structure than the WT FABP1 at all temperatures examined. For example, in the physiological temperature range AEA binding to FABP1 T94A variant increased the proportion of α-helix (Figure 10A) and β-sheet (Figure 10C), while decreasing that of turns (Figure 10E) and unordered structures (Figure 10G). Interestingly, at high temperatures (60-85oC) AEA binding to FABP1 T94A variant (but not to WT FABP1 T94T)

300

Ligand Induced Change in Secondary Structure (%) Unordered Turns β -Sheet α -Helix

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

AEA T94T T94A

A

200 100 0

-100 50

* *

*

400

2-AG

B

*

*

*

* **

300

*

* * * *

*

100 0

C

-100

D

*

* *

0

*

*

*

*

* 0

*

-50

-50

-100

E

*

-100

F

**

* *

0

*

* -50

-100

*

-40 -60

H

G

50

0

0

* * * *

* * * * *

-50

-150

40 20 0 -20

50

-100

200

*

*

* * 20

40

-100

* 60

80

20

40

-50

60

-150 80 100

o

Temperature, C Figure 10. Temperature effect of AEA and 2-AG binding on % change in secondary structure of human WT FABP1 and T94A variant. All conditions were as described in legend to Fig. 9 except data at each temperature point were expressed as the % change in each secondary structure element (FABP1/ligand – FABP1 only) of WT FABP1 T94T (solid black bars), and FABP1 T94A variant (open bars) upon interaction AEA (Panels A,C,E,G) or with 2-AG (Panels B,D,F,H). The ligand-induced % changes are shown for the proportion of α-helix (Panel A,B), β-sheet (Panel C,D), turns (Panels E,F), and unordered structure (Panels G,H). Values represent the mean ± SEM, n=6. *, P < 0.05 for T94A vs WT. 25 ACS Paragon Plus Environment

Biochemistry

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Page 26 of 38

dramatically increased the proportion of α-helix (Figure 10A) while decreasing that of unordered structures (Figure 10G). 2-AG binding was also effective in inducing more ordered structures in the FABP1 T94A variant protein than WT FABP1 T94T protein at almost all temperatures studied. In the 24-29oC range, 2-AG binding to WT FABP1 T94T increased the proportion of α-helix (Figure 10B), but not turns (Figure 10F) or β-sheet (Figure 10D), while decreasing unordered structures (Figure 10H). In the 24-29°C range AEA binding did not further impact the proportion of α-helix (Figure 10B), but decreased that of turns (Figure 10F) and β-sheet (Figure 10D) more, while increasing unordered structures (Figure 10H). AEA and 2AG binding to FABP1 T94A variant markedly altered this protein’s secondary structure at high temperature (Figure 10B,D,F,H), especially increasing the proportion of α-helix (Figure 10A-B).

DISCUSSION Free or bound EC levels within tissues are regulated not only by synthesis and degradation, but also by cytosolic binding/’chaperone’ proteins

49,53

.

Due to their poor aqueous solubility, ECs require

cytosolic binding proteins for transport/‘chaperoning’ from sites of synthesis/reuptake at the plasma membrane for targeting to intracellular sites such as endoplasmic reticulum (e.g. for degradation by FAAH) and nucleus (for activating peroxisome proliferator activated receptor-α, PPARα)

49

. Studies

with purified recombinant murine cytosolic lipidic ligand binding proteins have determined that several bind ECs with affinities in the following order: liver FABP1 FABP3,5, and 7)

49,50,54-57

1

> brain fatty acid binding proteins (i.e.

>> heat shock binding protein-70 (HSP70) 58. The first reports of a human

cytosolic EC binding protein found in brain showed that recombinant human sterol carrier protein-2 (SCP-2) bound AEA and 2-AG as well as transported bound AEA between model membranes

59,60

.

Although SCP-2 is also detected in liver, SCP-2’s concentration therein is nearly 10-fold lower than that of FABP1

60-64

and nearly half of SCP-2 is concentrated in liver peroxisomal matrix


65

. While murine

Page 27 of 38

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Biochemistry

FABP1 is the most prevalent cytosolic EC binding/‘chaperone’ protein in mouse liver 1, prior to the study presented herein almost nothing was known about the human FABP1 in this regard. Human FABP1 is the most highly prevalent lipidic (fibrate, bile acid, LCFA, LCFA-CoA, cholesterol) ligand binding protein present in human liver—accounting for 7-10% of cytosolic protein (700-1000 µM), several-fold more than observed in murine liver

19,20,22,47

. Since human FABP1 differs

significantly from murine FABP1 with respect to amino acid sequence, ligand binding cavity size, affinity for and structural response to binding of the above ligands

4,6,19,20,66

, findings of EC binding to murine

FABP1 may not readily be extrapolated to the human FABP1. Thus, almost nothing is known regarding: i) binding and specificity of human FABP1 for EC or EC analogues, ii) ability of EC or EC analogue binding to elicit conformational change in human FABP1, or iii) impact of the highly prevalent human FABP1 T94A variant on putative EC interactions. These issues were addressed herein with purified human recombinant WT FABP1 T94T (i.e. WT FABP1) and FABP1 T94A variant to provide the following new insights: First, human FABP1 was shown not only to bind EC, but that its EC binding site differed significantly from that of the murine as evidenced by a series of fluorescent ligand displacement assays. For example, human FABP1-bound cis-parinaroyl-CoA was not displaced by NAE—in marked contrast to rat WT FABP1-bound cis-parinaroyl-CoA which was readily displaced by nearly all NAE tested 1. Likewise, human FABP1-bound NBD-stearic acid was not displaced by NAE—again in contrast to rat FABP1-bound NBD-stearic acid which was displaced by multiple lipidic ligands FABP3,5, and 7-bound NBD-stearic acid which was readily displaced by NAE

49,50

44

and by murine

. Human FABP1-

bound DAUDA was displaced nearly 3-fold better by NAE (shown herein) than rat FABP1-bound DAUDA 1. In the case of ANS, neither human FABP1-bound ANS (shown herein) nor rat FABP1-bound ANS

1

were displaced by NAEs. Finally, spectral ‘blue’ shift analysis of NBD-AEA and NBD-2-AG

bound to human WT FABP1 and T94A variant indicated that these probes were localized within a relatively hydrophobic binding pocket, with that for NBD-AEA being localized/oriented within a slightly more polar region than NBD-2-AG in the WT FABP1, but not T94A variant binding pocket. 27 ACS Paragon Plus Environment

Taken

Biochemistry

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Page 28 of 38

together, the above studies with a variety of fluorescent probes suggest that the human FABP1 bound NAE and 2-MG, but the binding pocket of the human FABP1 differed significantly from that of the rat FABP1. Second, both direct NBD-AEA binding assay as well as its displacement by native NAE showed that human WT FABP1 and the T94A variant bound AEA with high affinity and 1:1 stoichiometry— analogous to their binding cholesterol with 1:1 stoichiometry 22. These findings were in marked contrast to human WT FABP1 (as well as T94A variant) each having two binding sites for a variety of non-EC ligands (fatty acid, fatty acyl-CoA, lysophospholipid, fibrate) 19,20,24. Further, NBD-AEA displacement by native NAEs showed that the human FABP1 WT and T94A bound NAEs with a wide range of affinities (OEA was significantly increased by the T94A substitution) with specificity in the order OEA > AEA, EPEA, DHEA. Generally, the human FABP1 WT and T94A variant bound NAEs typically nearly 10-fold more weakly than the rat FABP1.1 Taken together with the literature, these data indicated the following overall order of NAE affinities: rat FABP1

1

> human FABP1 T94A variant (shown herein) > human

WT FABP1 (shown herein), human SCP-2 1 >> heat shock binding protein-70 (HSP70) 58. The physiological significance of these findings lies in that human WT FABP1 is the most prevalent NAE binding protein present in human liver cytosol and may consequently regulate NAE levels therein. EC levels are regulated by tight control of synthesis and inactivation such that dysregulation thereof results in or is causative of pathological conditions such as obesity, metabolic syndrome, or neurological disorders

53

. For example, once NAEs (e.g. AEA) are released at the plasma membrane they remain

largely membrane associated because of their hydrophobicity

25

. Clearance of NAEs relies on cell

uptake/internalization and hydrolysis by the enzyme fatty acid amide hydrolase (FAAH) in the endoplasmic reticulum

25,67

. Consistent with this prediction, rat WT FABP1 enhances FAAH activity in

vitro 68 while FABP1 gene ablation increases mouse liver AEA level 1. Third, the human FABP1s appear unique among the FABP1s and/or other FABPs in selectively binding only one 2-MG, i.e. 2-AG. The NBD-2-AG displacement assay demonstrated that the human WT FABP1 bound 2-AG with nearly 7-fold higher affinity than for AEA. Uniquely, the human WT 28 ACS Paragon Plus Environment

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Biochemistry

FABP1 specifically bound only 2-AG, but not other 2-MGs tested (shown herein)—in marked contrast to the rat FABP1 which exhibited high affinity for 2-AG as well as 2-OG and 2-PG1. While the T94A substitution increased the human FABP1’s affinity for 2-AG nearly 2-fold, it still did not bind other 2MGs (shown herein). This places the human FABP1s in the following order of affinities among the known cytosolic 2-MG binding proteins: human T94A variant (shown herein), rat FABP1 1 > human WT FABP1 (shown herein), SCP-2 1. With regards to physiological significance of 2-AG binding, especially that of the human T94A variant, clearance of 2-MGs relies in part on cell uptake and hydrolysis by a different enzyme, i.e. monoacylglycerol lipase, which is primarily cytosolic 25,67. This suggests that human WT FABP1 binding of 2-AG may facilitate transfer/targeting to MGL for hydrolysis—thereby decreasing 2-AG levels. Consistent with this prediction, both human and rat WT FABP1 enhance MGL activity in vitro

68

and

FABP1 gene ablation increases mouse liver 2-AG level 1. Thus, the higher expression of total FABP1 in cultured primary human hepatocytes expressing the T94A variant would likely increase 2-AG hydrolysis to decrease tissue levels as was observed 1. Despite the T94A variant’s 2-fold higher affinity for 2-AG as compared to the human WT FABP1, the two recombinant proteins did not differ in ability to stimulate MGL in an in vitro assay 68. Fourth, the secondary structure/conformation of the human WT FABP1 protein is highly sensitive to EC binding. Binding of ECs altered human WT FABP1 secondary structure (especially distorted α-helix) to a greater extent (shown herein) than the impact of any other known FABP1 lipidic ligand (i.e. straightchain LCFA, branched-chain LCFA, LCFA-CoA, lysophosphatidic acid, phosphatidic acid, diglyceride, fibrates), regardless of species

19,20

. The ECs impact on human WT FABP1 secondary structure

conformation was markedly altered by the human FABP1 T94A substitution. The physiological significance of this finding relates to the impact of ligand-induced conformational change in FABP1 on downstream functions. For example, ligand (e.g. n-3 polyunsaturated fatty acid, fibrate)-induced conformational changes in FABP1 enhance cotransport of FABP1/ligand complex into nuclei

24,69

for interaction with/transfer of bound ligand to PPARα and activating its transcriptional 29 ACS Paragon Plus Environment

Biochemistry

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activity

7,35,36

Page 30 of 38

. Ablation of FABP1 completely abolishes while expression of the T94A variant markedly

diminishes the ability of such n-3-polyunsaturated fatty acids and fibrates to activate PPARα in cultured primary hepatocytes from mouse livers38,39,70 and human

19,20,24

. Similarly, NAEs such as OEA and PEA

are known to activate PPARα 71 and at higher concentration AEA binds to PPARγ 72. Murine FABP 3 and 7 are known to facilitate EC transport to PPARs

49,50

. Taken together these findings suggest that

diminution in human T94A variant of the conformational changes (normally induced in human WT FABP1) may adversely impact PPARα activation. Indeed, T94A variant expression induces triglyceride accumulation in cultured primary female human hepatocytes and NAFLD in human subjects

17,24

.

Furthermore, the elevated serum triglyceride level of human FABP T94A variant expressing subjects is less responsive to lowering to basal levels by fenofibrate 73. Fifth, in the absence of ligand, the T94A variant was more sensitive to temperature induced unfolding—consistent with an earlier finding 19. 2-AG and AEA partially stabilized the structure of the T94A variant to temperature-induced unfolding and less so the WT FABP1 to temperature-induced unfolding, mostly in α-helix and β-sheet, and unordered. In fact, AEA and 2-AG almost made the two proteins indistinguishable in thermal stability across the tested range. Surprisingly, the largest changes occurred at higher temperatures in the T94A variant bound to ECs principally within its α-helix. The moderate stabilizing effect of AEA and 2-AG binding on the ability of WT FABP1 to unfolding conditions (i.e. as exemplified at increased temperature) may significantly contribute to the functional interaction of WT FABP1 with other proteins such as degradative enzymes (FAAH, MAGL) for effectively transferring bound AEA or 2-AG. This would be analogous to fatty acyl-CoA binding to murine FABP1 altering its conformation to facilitate its interaction with the outer mitochondrial membrane CPT1 (rate limiting in mitochondrial fatty acid oxidation) to facilitate fatty acyl CoA transfer/transacylation and mitochondrial oxidation 34. Conversely, the much higher effect of AEA and 2AG binding on stabilizing the FABP1 T94A variant to unfolding conditions may preclude its ability to effectively interact with and transfer bound AEA or 2-AG to degradative enzymes (FAAH, MAGL). This


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Biochemistry

would be analogous to mutations in CPT1A that inhibit interaction with and conformational change induced by FABP1 binding—thereby preventing effective transfer of bound fatty acyl-CoA and thus inhibit mitochondrial oxidation

34

. Further studies beyond the scope of the present investigation will

examine the impact of the T94A variant on these and other functional interactions of the human FABP1 with individual components of the EC system.

CONCLUSIONS Human WT FABP1 protein differed significantly from that of other species (e.g. rat FABP1) in quantitative pattern of affinities for EC as well as structural responsiveness to EC binding. EC binding affinity/specificity as well as EC binding-induced structural alterations obtained for the only other known species examined (i.e. rat FABP1) may not necessarily be readily extrapolated to that of the human WT FABP1. The contrasting binding affinities for NAE and 2-MG, deduced for human and rat FABP1’s from fluorescent ligand displacement assays, suggest differences in the binding pockets for the two proteins, although these assays do not provide further molecular detail. Nonetheless, the results demonstrate localization of the NBD probes within hydrophobic regions and 1:1 stoichiometry that is specific to EC’s as compared with other hydrophobic ligands. The ability of the human FABP1 to bind only 2-AG among the 2-MGs is also a physiologically interesting finding, because it suggests a mechanism to target this ligand for hydrolysis by a specific hydrolase enzyme. In general the impact of the T94A variant was modest in terms of EC binding affinities and contrasted with prior expectations of broader blockage of ligand binding 23. The finding of diminished sensitivity of protein secondary structure to EC binding has possible significance in terms of PPARα activation. Both sets of observations for FABP1 binding lay the groundwork for more ambitious studies of how these binding phenomena play out in vivo and/or can be rationalized at the level of molecular structure.

Acknowledgements This work was supported in part by TxAgriLife Research Stimulus Funds (FS). 31 ACS Paragon Plus Environment

Biochemistry

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Notes The authors have no conflicts of interest with this article’s contents, which is solely the responsibility of the authors and does not necessarily represent the official views of TxAgriLife.

REFERENCES

1. Huang, H., McIntosh, A. L., Martin, G. G., Landrock D., Chung, S., Landrock, K. K., Dangott, L. J., Li, S., Kier, A. B., and Schroeder, F. (2016) FABP1: a novel hepatic endocannabinoid and cannabinoid binding protein., Biochemistry 55, 5243-5255. 2. Lowe, J. B., Boguski, M. S., Sweetser, D. A., Elshourbagy, N., Taylor, J. M., and Gordon, J. I. (1985) Human liver fatty acid binding protein: Isolation of a full length cDNA and comparative sequence analyses of orthologous and paralogous proteins., J. Biol. Chem. 260, 3413-3417. 3. Chan, L., Wei, C. F., Li, W. H., Yang, C. Y., Ratner, P., Pownall, H., Gotto, A. M., Jr., and Smith, L. C. (1985) Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications, J. Biol. Chem. 260, 2629-2632. 4. Maatman, R. G., van de Westerlo, E. M., Van Kuppevelt, T. H., and Veerkamp, J. H. (1992) Molecular identification of the liver- and the heart-type fatty acid-binding proteins in human and rat kidney. Use of the reverse transcriptase polymerase chain reaction, Biochem. J. 288, 285-290. 5. Maatman, R. G., Degano, M., van Moerkerk, H. T., Van Marrewijk, W. J., Van der Horst, D. J., Sacchettini, J. C., and Veerkamp, J. H. (1994) Primary structure and binding characteristics of locust and human muscle fatty-acid-binding proteins, Eur. J. Biochem. 221, 801-810. 6. Wolfrum, C., Borchers, T., Sacchettini, J. C., and Spener, F. (2000) Binding of fatty acids and perooxisome proliferators to orthologous fatty acid binding proteins from human, murine, and bovine liver., Biochemistry 39, 1469-1474. 7. Velkov, T. (2013) Interactions between human liver fatty acid binding protein and peroxisome proliferator activated receptor drugs., PPAR Research 2013, 1-14. 8. Long, D. and Yang, D. (2011) Millisecond timescale dynamics of human liver fatty acid binding protein: testing of its relevance to the ligand entry process., Biophys. J. 98, 3054-3061. 9. Long, D. and Yang, D. (2009) Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein., Biophys. J. 96, 1482-1488. 10. Sharma, A. and Sharma, A. (2011) Fatty acid induced remodeling within the human liver fatty acid binding protein., J. Bio. Chem. 286, 31924-31928. 11. Cai, J., Lucke, C., Chen, Z., Qiao, Y., Klimtchuk, E., and Hamilton, J. A. (2012) Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited., Biophys. J. 102, 2585-2594. 32 ACS Paragon Plus Environment

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

12. Robitaille, J., Brouillette, C., Lemieux, S., Perusse, L., Gaudet, D., and Vohl, M.-C. (2004) Plasma concentrations of apolipoprotein B are modulated by a gene-diet interaction effect between the LFABP T94A polymorphism and dietary fat intake in French-Canadian men., Mol. Gen. and Metab. 82, 296-303. 13. Fisher, E., Weikert, C., Klapper, M., Lindner, I., Mohlig, M., Spranger, J., Boeing, H., Schrezenmeir, J., and Doring, F. (2007) L-FABP T94A is associated with fasting triglycerides and LDLcholesterol in women., Mol. Gen. and Metab. 91, 278-284. 14. Weikert, M. O., Loeffelholz, C. v., Roden, M., Chandramouli, V., Brehm, A., Nowotny, P., Osterhoff, M. A., Isken, F., Spranger, J., Landau, B. R., Pfeiffer, A., and Mohlig, M. (2007) A Thr94Ala mutation in human liver fatty acid binding protein contributes to reduced hepatic glycogenolysis and blunted elevation of plasma glucsoe levels in lipid-exposed subjects., Am. J. Physiol. Endocrinol. Metab. 293, E1078-E1084. 15. Yamada, Y., Kato, K., Oguri, M., Yoshida, T., Yokoi, K., Watanabe, S., Metoki, N., Yoshida, H., Satoh, K., Ichihara, S., Aoyagi, Y., Yasunaga, A., Park, H., Tanaka, M., and Nozawa, Y. (2008) Association of genetic variants with atherothrombotic cerebral infarction in Japanese individuals with metabolic syndrome., Int. J. Mol. Med. 21, 801-808. 16. Bu, L., Salto, L. M., De Leon, K. J., and De Leon, M. (2011) Polymorphisms in fatty acid binding protein 5 show association with type 2 diabetes., Diabetes Res. Clin. Prac. 92, 82-91. 17. Peng, X.-E., Wu, Y. L., Lu, Q.-Q., Ju, Z.-J., and Lin, X. (2012) Two genetic variants in FABP1 and susceptibility to non-alcoholic fatty liver disease in a Chinese population., Gene 500, 54-58. 18. Mansego, M. L., Martinez, F., Martinez-Larrad, M. T., Zabena, C., Rojo, G., Morcillo, S., Soriguer, F., Martin-Escudero, J. C., Serrano-Rios, M., Redon, J., and Chaves, F. J. (2012) Common variants of the liver fatty acid binding protein gene influence the risk of Type 2 Diabetes and insulin resistance in Spanish population., PLoS ONE 7, e31853. 19. Martin, G. G., McIntosh, A. L., Huang, H., Gupta, S., Atshaves, B. P., Kier, A. B., and Schroeder, F. (2013) Human liver fatty acid binding protein (L-FABP) T94A variant alters structure, stability, and interaction with fibrates., Biochemistry 52, 9347-9357. 20. Huang, H., McIntosh, A. L., Martin, G. G., Landrock, K., Landrock, D., Gupta, S., Atshaves, B. P., Kier, A. B., and Schroeder, F. (2014) Structural and functional interaction of fatty acids with human liver fatty acid binding protein (L-FABP) T94A variant., FEBS J. 281, 2266-2283. 21. D'Onofrio, M., Barracchia, C. G., Bortot, A., Munari, F., Zanzoni, S., and Assfalg, M. (2017) Molecular differences between human liver fatty acid binding protein and its T94A variant in their unbound and lipid bound states., Biochim. Biophys. Acta-Proteins and Proteomics 1865, 1152-1159. 22. Huang, H., McIntosh, A. L., Martin, G. G., Landrock, K. K., Landrock, D., Storey, S. M., Gupta, S., Atshaves, B. P., Kier, A. B., and Schroeder, F. (2015) Human L-FABP T94A variant enhances cholesterol uptake., Biochim. Biophys. Acta 1851, 946-955. 23. Gao, N., Qu, X., Yan, J., Huang, Q., Yuan, H. Y., and Ouyang, D.-S. (2010) L-FABP T94A decreased fatty acid uptake and altered hepatic triglyceride and cholesterol accumulation in Chang liver cells stably transfected with L-FABP., Mol. Cell. Biochem. 345, 207-214. 33 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

24. McIntosh, A. L., Huang, H., Storey, S. M., Landrock, K., Landrock D., Petrescu, A. D., Gupta, S., Atshaves, B. P., Kier, A. B., and Schroeder, F. (2014) Human FABP1 T94A variant impacts fatty acid metabolism and PPARa activation in cultured human female hepatocytes., Am. J. Physiol. Gastrointest. and Liver Phys. 307, G164-G176. 25. Alswat, K. A. (2015) The role of endocannabinoid system in fatty liver disease and therapeutic potential., Saudi Journal of Gastroenterology 19, 144-151. 26. Tam, J., Liu, J., Mukhopadhyay, B., Cinar, R., Godlewski, G., and Kunos, G. (2011) Endocannabinoids in liver disease., Hepatology 53, 346-355. 27. Regnell, S. E. (2013) Cannabinoid 1 receptor in fatty liver., Hepatol. Res. 43, 131-138. 28. Yang, S. Y., He, X. Y., and Schulz, H. (1987) Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase, J. Biol. Chem. 262, 13027-13032. 29. Higuchi, N., Kato, M., Tanaka, M., and et al (2011) Effects of insulin resistance and hepatic lipid accumulation on hepatic mRNA expression levels of apoB, MTP, and L-FABP in non-alcoholic fatty liver disease., Exp. and Ther. Med. 2, 1077-1081. 30. Charlton, M., Viker, K., Krishnan, A., Sanderson, S., Veldt, B., Kaalsbeek, A. J., Kendrick, M., Thompson, G., Que, F., Swain, J., and Sarr, M. (2009) Differential expression of lumican and fatty acid binding protein-1: new insights into the histologic spectrum of nonalcoholic fatty liver disease., Hepatology 49, 1375-1384. 31. Baumgardner, J. N., Shankar, K., Hennings, L., Badger, T. M., and Ronis, M. J. J. (2007) A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a highpolyunsaturated fat diet., Am. J. Physiol. Gastrointest. and Liver Phys. 294, G27-G38. 32. Osei-Hyiaman, D., Liu, J., Zhou, L., Godlewski, G., Harvey-White, J., Jeong, W., Batkai, S., Marsicano, G., Lutz, B., Buettner, C., and Kunos, G. (2008) Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice., J. Clin. Inv. 118, 3160-3169. 33. Storey, S. M., McIntosh, A. L., Huang, H., Martin, G. G., Landrock, K. K., Landrock, D., Payne, H. R., Kier, A. B., and Schroeder, F. (2012) Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes., Am. J. Physiol. Gastrointest. and Liver Phys. 303, G837-G850. 34. Hostetler, H. A., Lupas, D., Tan, Y., Dai, J., Kelzer, M. S., Martin, G. G., Woldegiorgis, G., Kier, A. B., and Schroeder, F. (2011) Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyltransferase I., Mol. Cell. Biochem. 355, 135-148. 35. Hostetler, H. A., McIntosh, A. L., Atshaves, B. P., Storey, S. M., Payne, H. R., Kier, A. B., and Schroeder, F. (2009) Liver type Fatty Acid Binding Protein (L-FABP) interacts with peroxisome proliferator activated receptor-a in cultured primary hepatocytes., J. Lipid Res. 50, 1663-1675. 36. Hostetler, H. A., Balanarasimha, M., Huang, H., Kelzer, M. S., Kaliappan, A., Kier, A. B., and Schroeder, F. (2010) Glucose regulates fatty acid binding protein interaction with lipids and PPARa, J. Lipid Res. 51, 3103-3116.


Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

37. Schroeder, F., Petrescu, A. D., Huang, H., Atshaves, B. P., McIntosh, A. L., Martin, G. G., Hostetler, H. A., Vespa, A., Landrock, K., Landrock, D., Payne, H. R., and Kier, A. B. (2008) Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription., Lipids 43, 1-17. 38. Petrescu, A. D., McIntosh, A. L., Storey, S. M., Huang, H., Martin, G. G., Landrock, D., Kier, A. B., and Schroeder, F. (2013) High glucose potentiates liver fatty acid binding protein (L-FABP) mediated fibrate induction of PPARa in mouse hepatocytes., Biochim. Biophys. Acta 1831, 14121425. 39. Petrescu, A. D., Huang, H., Martin, G. G., McIntosh, A. L., Storey, S. M., Landrock, D., Kier, A. B., and Schroeder, F. (2013) Impact of L-FABP and glucose on polyunsaturated fatty acid induction of PPARa regulated b-oxidative enzymes., Am. J. Physiol. Gastrointest. and Liver Phys. 304, G241-G256. 40. Huang, H., Atshaves, B. P., Frolov, A., Kier, A. B., and Schroeder, F. (2005) Acyl-coenzyme A binding protein expression alters liver fatty acyl coenzyme A metabolism., Biochemistry 44, 10282-10297. 41. Martin, G. G., Huang, H., Atshaves, B. P., Binas, B., and Schroeder, F. (2003) Ablation of the liver fatty acid binding protein gene decreases fatty acyl CoA binding capacity and alters fatty acyl CoA pool distribution in mouse liver., Biochem. 42, 11520-11532. 42. Frolov, A., Cho, T. H., Billheimer, J. T., and Schroeder, F. (1996) Sterol carrier protein-2, a new fatty acyl coenzyme A-binding protein, J. Biol. Chem. 271, 31878-31884. 43. Thumser, A. E., Voysey, J. E., and Wilton, D. C. (1994) The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin, Biochem. J. 301, 801-806. 44. Schroeder, F., Myers-Payne, S. C., Billheimer, J. T., and Wood, W. G. (1995) Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol, Biochemistry 34, 1191911927. 45. Martin, G. G., Atshaves, B. P., Huang, H., McIntosh, A. L., Williams, B. W., Pai, P.-J., Russell, D. H., Kier, A. B., and Schroeder, F. (2009) Hepatic phenotype of liver fatty acid binding protein (LFABP) gene ablated mice., Am. J. Physiol. 297, G1053-G1065. 46. Carbone, V. and Velkov, T. (2013) Interaction of phthalates and phonxy acid herbicide environmental pollutants with intestinal intracellular lipid binding proteins., Chem. Res. in Tox. 26, 1240-1250. 47. Favretto, F., Assfalg, M., Gallo, M., Cicero, D. O., D'Onofrio, M., and Molinari, H. (2013) Ligand binding promiscuity and human liver fatty acid binding protein: structural and dynamic insights from an interaction study with glycocholate and oleate., ChemBioChem 14, 1807-1819. 48. Santambrogio, C., Favretto, F., D'Onofrio, M., Assfalg, M., Grandori, R., and Molinari, H. (2013) Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein., J. Mass Spectrometry 48, 895-903. 49. Kaczocha, LM., Glaser, S. T., and Deutsch, D. G. (2009) Identification of intracellular carriers for the endocannabinoid anandamide., Proc. Natl. Acad. Sci. U. S. A. 106, 6375-6380. 35 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

50. Kaczocha, M., Vivieca, S., Sun, J., Glaser, S. T., and Deutsch, D. G. (2012) Fatty acid binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors., J. Biol. Chem. 287, 3415-3424. 51. Schroeder, F., McIntosh, A. L., Martin, G. G., Huang, H., Landrock D., Chung, S., Landrock, K. K., Dangott, L. J., Li, S., Kaczocha, M., Murphy, E. J., Atshaves, B. P., and Kier, A. B. (2016) Fatty acid binding protein-1 (FABP1) and the human FABP1 T94A variant: Roles in the endocannabinoid system and dyslipidemias., Lipids 51, 655-676. 52. McIntosh, A. L., Atshaves, B. P., Hostetler, H. A., Huang, H., Davis, J., Lyuksyutova, O. I., Landrock, D., Kier, A. B., and Schroeder, F. (2009) Liver type fatty acid binding protein (LFABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferator activated receptor-alpha activity in cultured primary hepatocytes., Arch. Biochem. Biophys. 485, 160-173. 53. Muccioli, G. G. (2010) Endocannabinoid biosynthesis and inactivation, from simple to complex., Drug Discovery Today 15, 474-483. 54. Berger, W. T., Ralph, B. P., Kaczocha, M., Sun, J., Balius, T. E., Rizzo, R. C., Haj-Dahmane, S., Ojima, I., and Deutsch, D. G. (2012) Targeting FABP anandamide transporters--a novel strategy for development of anti-inflammatory and anti-nociceptive drugs., PLoS ONE 7, e50968. 55. Kaczocha, M. (2009) Role of fatty acid binding proteins and FAAH-2 in endocannabinoid uptake and inactivation., Ph. D. Thesis, Stony Brook University. 56. Kaczocha, M., Rebecchi, M. J., Ralph, B. P., Teng, Y.-H. G., Berger, W. T., Galbavy, W., Elmes, M. W., Glaser, S. T., Wang, L., Rizzo, R. C., Deutsch, D. G., and Ojima, I. (2014) Inhibition of fatty acid binding protein elevates brain anandamide levels and produces analgesia., PLoS ONE 9, e94200. 57. Kaczocha, M., Glaser, S. T., Maher, T., Clavin, B., Hamilton, J., O'Rourke, J., Rebecchi, M., Puopolo, M., Owada, Y., and Thanos, P. K. (2015) Fatty acid binding protein deletion suppresses inflammatory pain through endocannabinoid/N-acylethanolamine-dependent mechanisms., Molecular Pain 11, 52. 58. Oddi, S., Fezza, F., Pasquoriello, N., and et al (2009) Molecular identification of albumin and Hsp70 as cytosolic anandamide binding proteins., Chemistry and Biology 16, 624-632. 59. Liedhegner, E. S., Vogt, C. D., Sem, D. S., Cunninham, C. W., and Hillard, C. J. (2014) Sterol carrier protein-2: binding protein for endocannabinoids., Mol. Neurobiol. 50, 149-158. 60. Martin, G., Chung, S., Landrock D., Landrock, K. K., Huang, H., Dangott, L. J., Peng, X., Kaczocha, M., Seeger, D. R., Murphy, E. J., Golovko, M. Y., Kier, A. B., and Schroeder, F. (2016) FABP1 gene ablation impacts brain endocannabinoid system in male mice., J. Neurochem. 138, 407-422. 61. McArthur, M. J., Atshaves, B. P., Frolov, A., Foxworth, W. D., Kier, A. B., and Schroeder, F. (1999) Cellular uptake and intracellular trafficking of long chain fatty acids, J. Lipid Res. 40, 1371-1383. 62. Atshaves, B. P., Martin, G. G., Hostetler, H. A., McIntosh, A. L., Kier, A. B., and Schroeder, F. (2010) Liver fatty acid binding protein (L-FABP) and Dietary Obesity., Journal of Nutritional Biochemisty 21, 1015-1032.


Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

63. Schroeder, F., Frolov, A., Schoer, J., Gallegos, A., Atshaves, B. P., Stolowich, N. J., Scott, A. I., and Kier, A. B. (1998) Intracellular sterol binding proteins, cholesterol transport and membrane domains, in Intracellular Cholesterol Trafficking (Chang, T. Y. and Freeman, D. A., Eds.,) pp 213-234, Kluwer Academic Publishers, Boston. 64. Gossett, R. E., Frolov, A. A., Roths, J. B., Behnke, W. D., Kier, A. B., and Schroeder, F. (1996) Acyl Co A binding proteins: multiplicity and function., Lipids 31, 895-918. 65. Gallegos, A. M., Atshaves, B. P., Storey, S. M., Starodub, O., Petrescu, A. D., Huang, H., McIntosh, A., Martin, G., Chao, H., Kier, A. B., and Schroeder, F. (2001) Gene structure, intracellular localization, and functional roles of sterol carrier protein-2., Prog. Lipid Res. 40, 498-563. 66. Richieri, G. V., Ogata, R. T., and Kleinfeld, A. M. (1994) Equilibrium constants for the binding of fatty acids with fatty acid binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB., J. Biol. Chem. 269, 23918-23930. 67. Mallat, A. and Lotersztain, S. (2008) Endocannabinoids and liver disease. I. Endocannabinoids and their receptors in liver., Am. J. Physiol. Liver Physiol. 294, G9-G12. 68. McIntosh, A. L., Huang, H., Landrock.D., Li, S., Kier, A. B., and Schroeder, F. (2017) Novel NBDprobes for endocannabinoid imaging and metabolism in hepatocytes: Impact of Fabp1 gene ablation., Biochim. Biophys. Acta - Mol. Cell Biol. Lip. Submitted 2-17-17. 69. Lawrence, J. W., Kroll, D. J., and Eacho, P. I. (2000) Ligand dependent interaction of hepatic fatty acid binding protein with the nucleus., J. Lipid Res. 41, 1390-1401. 70. Huang, H., McIntosh, A. L., Martin, G. G., Petrescu, A. D., Landrock, K., Landrock, D., Kier, A. B., and Schroeder, F. (2013) Inhibitors of fatty acid synthesis induce PPARa-regulated fatty acid boxidative enzymes: synergistic roles of L-FABP and glucose., PPAR Research 2013, 1-22. 71. Silvestri, C. and DiMarzo, V. (2013) The endocannabinoid system in energy homeostasis and the etiophathology of metabolic disorders., Cell Metabolism 17, 475-490. 72. Guzman, M., Lo Verme, J., Fu, J., Oveisi, F., Blazquez, C., and Piomelli, D. (2004) Oleoylethanolamide stimulates lipolysis by activating the nuclear receptor PPARa., J. Biol. Chem. 279, 27849-27854. 73. Brouillette, C., Bose, Y., Perusse, L., Gaudet, D., and Vohl, M.-C. (2004) Effect of liver fatty acid binding protein (FABP) T94A missense mutation on plasma lipoprotein responsiveness to treatment with fenofibrate., J. Hum. Gen. 49, 424-432.


Biochemistry

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Endocannabinoid Interaction with Human FABP1: Impact of T94A Variant Gregory G. Martin*, Huan Huang*, Avery L. McIntosh*, Ann B. Kier‡, and Friedhelm Schroeder*,1


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Endocannabinoid Interaction with Human FABP1: Impact of the T94A

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