Human Fatty Acid Binding Protein Research Articles

Basic Fibroblast Growth Factor and Epithelial Growth Factor could Induce Trans-differentiation of Mesenchymal Stem Cell into Neural Stem Cell

Background & Aim Background Stem cell therapy has been considered as a potential strategy in regenerative for the patient with neurological deficits. Mesenchymal Stem/Stromal Cells (MSC) have been extensively investigated as a treatment in stroke. However, the MSC lost its potency for neural repair in aged patients due to the limited numbers of Neural Stem Cell (NSC). NSC is essential for neurogenesis of the injured brain. In vitro studies have shown that MSCs which were cultured in the laboratory has the capability to transform into different cells. Adding stimulants such as growth factors into the culture media could induce the trans-differentiation of MSC into NSC. However, to produce NSC in the clinical setting on a large scale is still a challenge. Objective In this study, we like to evaluate the use of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heregulin B27, and N2 the trans-differentiation mechanism of umbilical cord MSC (UC-MSC) into NSC. Methods, Results & Conclusion Method MSC was transferred into a 24-well plate at 2 × 104 cells per well. Induction of NSC is done using DMEM containing N2/B27 supplement, 20 ng/ml epidermal growth factor (EGF), and 10 ng/ml basic fibroblast growth factor (bFGF). Identification of success trans-differentiation is analyzed using NeuroFlour CDr3 antibody. Result and Discussion MSC transdifferentiates into NSC as an important source of cell in therapy for neurological diseases. UC-MSC was induced using N2 to helps the cell to commit to differentiation and its survival. B27 is used for the long term survival of neurons cells and helps the NSC to grow and proliferates. bFGF beliefs as pre-inductive molecules that enhance neuronal differentiation and self-renewal. Upon bFGF stimulation, NGFβR and NRP1 will upregulate and promotes differentiation. On the contrary, EGF induces differentiation at lower concentrations. EGF is used for inducing the expression of the neuron and glial marker. To confirm the successful transdifferentiating process, the expression of CDr3 in the cell culture is analyzed. CDr3 positive result as the membrane-permeable fluorescent probe was shown in Figure 1. NeuroFluor CDr3 binds to human fatty acid-binding protein 7 (FABP7) which use to confirm the neural induction from human stem cells. Conclusion UC-MSC could be cultured and differentiate into NSC in vitro when supplemented with N2/B27, bFGF, and EGF, which may produce NSC in the clinical setting on a large scale. Stem cell therapy has been considered as a potential strategy in regenerative for the patient with neurological deficits. Mesenchymal Stem/Stromal Cells (MSC) have been extensively investigated as a treatment in stroke. However, the MSC lost its potency for neural repair in aged patients due to the limited numbers of Neural Stem Cell (NSC). NSC is essential for neurogenesis of the injured brain. In vitro studies have shown that MSCs which were cultured in the laboratory has the capability to transform into different cells. Adding stimulants such as growth factors into the culture media could induce the trans-differentiation of MSC into NSC. However, to produce NSC in the clinical setting on a large scale is still a challenge. In this study, we like to evaluate the use of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heregulin B27, and N2 the trans-differentiation mechanism of umbilical cord MSC (UC-MSC) into NSC. MSC was transferred into a 24-well plate at 2 × 104 cells per well. Induction of NSC is done using DMEM containing N2/B27 supplement, 20 ng/ml epidermal growth factor (EGF), and 10 ng/ml basic fibroblast growth factor (bFGF). Identification of success trans-differentiation is analyzed using NeuroFlour CDr3 antibody.

Nontarget Screening of Per- and Polyfluoroalkyl Substances Binding to Human Liver Fatty Acid Binding Protein.

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein (hL-FABP). Of 74 PFASs assessed, 20 were identified as hL-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI-) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify hL-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new hL-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C8- and C10/EOn, C8H17(C2H4O)nSO4-, and C10H21(C2H4O)nSO4-). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C14- and C15/EOn. This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown hL-FABP ligands.

The role of human heart-type Fatty Acid Binding Protein in the early detection of myocardial injury in Acute Coronary Syndrome

Objectives: To evaluate the use of H-FABP as a novel marker in early detection of cardiac damage (MI) in patients presenting with symptoms of acute coronary syndrome (ACS). Methods: This cross-sectional study included 250 subjects; 125 patients of ACS presenting within four hours of the onset of chest pain and/dyspnea and 125 age and sex-matched healthy controls. An initial blood sample was taken from patients at presentation. Blood samples of healthy control subjects were also taken. The blood samples of both groups were centrifuged and stored at -200 C for H-FABP analysis. All the patients and control subjects were thoroughly examined and detailed history was taken. The diagnostic test was troponin-T.H-FABP concentrations of all samples were measured by ELISA-kit. The results were analyzed statistically. A p-value≤0.05 was considered statistically significant. Results: In 125 patients of ACS, H-FABP showed a sensitivity of 85% and specificity of 83.3% for acute MI diagnosis at a cut-off level of 16 ng/ml. Positive predictive values and negative predictive values were 96.8% and 48.4% respectively. The accuracy was 84.4%and the area under the ROC curve was 0.842. Conclusion: Evaluation of heart-type fatty acid-binding Protein (H-FABP) within four hours of onset ACS symptoms may be a valuable tool in the diagnosis of AMI. Keywords: Heart-type fatty acid-binding protein, Acute coronary syndrome, Myocardial infarction.

A NMR-based drug screening strategy for discovering active substances from herbal medicines: Using Radix Polygoni Multiflori as example

Ethnopharmacological relevanceHerbal medicines have always been important sources for new drugs. And developing new drugs from traditional herbal medicine is currently still an effective way. However, screening for active substances from herbal medicines extracts has ever been a challenging topic, due to their intrinsic complexity.The herb Radix Polygoni Multiflori has been used as a tonic and an antiaging herb in Traditional Chinese Medicine. In clinical studies, the extract of Radix Polygoni Multiflori can improve hypercholesterolemia, atherosclerotic, diabetes and other diseases commonly associated with glycolipid metabolism, however, the molecular mechanisms of these actions are unknown. Aim of the studyWe devised a NMR-based drug screening strategy for discovering active substances from herbal medicines, using Radix Polygoni Multiflori as example to address such challenging topic, meanwhile, to explore molecular target of Radix Polygoni Multiflori's glycolipid metabolism benefit. Materials and methodsHerbal medicines extracts were subjected to moderate separation to generate libraries of pre-purified subfractions, target protein was then added to each subfraction, and ligand-observed NMR experiments (line-broadening experiment, chemical shift perturbations measurements and saturation transfer difference spectrum) were performed, active substances identification and structural optimization were then accomplished using signals provided by ligand-observed NMR interaction detection and HPLC-SPE-NMR. The strategy was demonstrated by discovering an active component from extract of herb Radix Polygoni Multiflori, using human fatty acid binding protein 4 (FABP4) as target protein. Results2,4-dihydroxy-6-[(1E)-2-(4-hydroxyphenyl)ethenyl]phenyl-ß-D-glucopyranoside(TSG), the hit from one subfraction, has obvious interaction with target protein FABP4, due to FABP4 is a potential therapeutic target for metabolic diseases such as diabetes and atherosclerosis, the screening result will give clue to the active component and molecular target of Radix Polygoni Multiflori's glycolipid metabolism benefit. Besides, interaction information at atom level offered by ligand-observed NMR experiment would be valuable in the further stage of lead optimization. ConclusionsThe devised NMR-based drug screening strategy can discover active substances from herbal medicines efficiently and precisely, meanwhile, can shed light on molecular mechanism of traditional usage of the herb.

Tissue distribution and bioaccumulation of a novel polyfluoroalkyl benzenesulfonate in crucian carp

The emergence of novel per- and polyfluoroalkyl substances (PFASs) has enabled researchers to determine their bioaccumulation, which is important for understanding their internal doses and environmental risks. Here, for the first time, we report on the occurrence of a novel PFAS, p-perfluorous nonenoxybenzenesulfonate (OBS) in wild crucian carp and explore its tissue distribution and bioaccumulation. The highest levels of OBS were observed in blood (mean/median: 144/133 ng/ml) with the mean tissue/blood ratios (TBRs) consistently below 1, ranging from 0.090 (muscle) to 0.644 (liver). This followed the pattern of perfluorooctane sulfonate (PFOS), implying that their distributions were similar. The calculated tissue-specific LogBAF values, except for muscle, 3.78 (gill)–4.14 (blood) over the regulatory bioaccumulation criterion (Log value: 3.70) indicated its obvious bioaccumulative potency in crucian carp. Molecular docking with estimated binding energies at −8.5 and −9.0 kcal/mol corroborated the strong interactions of OBS with human serum albumin and liver fatty acid binding protein, even though the binding energies were lower than those of PFOS. This, to some extent, explained the lower bioaccumulation of OBS than PFOS. Considering its bioaccumulative potential, large production volume, and wide use, further investigation into the environmental risk and in vivo toxicology of OBS is required.

Ligand Entry into Fatty Acid Binding Protein via Local Unfolding Instead of Gap Widening

Fatty acid binding proteins play an important role in the transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still takes up fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer, and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like states, and two locally unfolded states in aqueous solution. Local unfolding of either βB–βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.

Identification of B Cell Epitopes of Blo t 13 Allergen and Cross-Reactivity with Human Adipocytes and Heart Fatty Acid Binding Proteins.

Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.

Increased ileal bile acid binding protein and galectin-9 are associated with mild cognitive impairment and Alzheimer's disease

Enterocyte damage and subsequent microbial translocation drive neuroinflammation in the pathogenesis of Alzheimer's disease (AD). Human ileal bile acid binding protein (I-BABP) and intestinal fatty acid binding proteins (I-FABP) are the indicators of enterocyte damage. Lipopolysaccharide-binding protein (LBP) is an indirect marker of microbial translocation. The activation of peripheral innate immune cells plays a crucial role in modulating AD progression. Galectin-9 is a versatile immunomodulatory molecule. The purpose of this study was to determine I-FABP, I-BABP, LBP, and galectin-9 levels in MCI and AD and investigate the relationship between I-FABP, I-BABP, LBP and galectin-9. In this study, I-FABP, I-BABP, LBP, and galectin-9 levels were measured using ELISA assay in 115 AD patients, 115 MCI patients, and 115 non-demented control subjects. Increased I-BABP and galectin-9 were observed in MCI and AD patients. Furthermore, AD patients had higher I-BABP and galectin-9 levels compared with MCI patients. However, I-FABP and LBP in three groups had no difference. I-BABP levels were positively correlated with galectin-9, after adjusting confounding factors (r = 0.409, p < 0.001). In addition, multivariate analysis revealed that increased I-BABP and galectin-9 levels were significantly associated with reduced mini-mental state examination (MMSE) score. In conclusion, galectin-9 is correlated with I-BABP after adjusting confounding covariates. Moreover, increased I-BABP and galectin-9 in MCI and AD are significant factors for reduced MMSE score. Further studies are needed.

Signal-Enhanced Detection of Multiplexed Cardiac Biomarkers by a Paper-Based Fluorogenic Immunodevice Integrated with Zinc Oxide Nanowires.

Using a single test to comprehensively evaluate multiple cardiac biomarkers for early diagnosis and prevention of acute myocardial infarction (AMI) has faced enormous challenges. Here, we have developed paper-based fluorogenic immunodevices for multiplexed detection of three cardiac biomarkers, namely, human heart-type fatty acid binding protein (FABP), cardiac troponin I (cTnI), and myoglobin, simultaneously. The detection is based on a strategy using zinc oxide nanowires (ZnO NWs) to enhance fluorescence signals (∼5-fold compared to that on pure paper). The immunodevices showed high sensitivity and selectivity for FABP, cTnI, and myoglobin with detection limits of 1.36 ng/mL, 1.00 ng/mL, and 2.38 ng/mL, respectively. Additionally, the paper-based immunoassay was rapid (∼5 min to complete the test) and portable (using a homemade chamber with a smartphone and an ultraviolet lamp). The developed devices integrated with ZnO NWs enable quantitative, sensitive, and simultaneous detection of multiple cardiac biomarkers in point-of-care settings, which provides a useful approach for monitoring AMI diseases and may be extended to other medical diagnostics and environmental assessments.

Renoprotective effects of voluntary running exercise training on aldosterone-induced renal injury in human L-FABP chromosomal transgenic mice.

Tubulointerstitial damage is a crucial therapeutic target in preventing chronic kidney disease (CKD) progression. Inappropriately activated renin-angiotensin-aldosterone system (RAAS) in the tubulointerstitial area is strongly associated with tubulointerstitial damage progression. Therefore, this study aimed to determine whether there is a beneficial effect of voluntary running exercise training on aldosterone-induced renal injury. Human L-type fatty acid-binding protein (L-FABP) chromosomal transgenic (L-FABP+/-) male mice were used to evaluate the effect of exercise by using urinary L-FABP, a tubular marker. The mice were assigned to either the exercise group that performed voluntary running exercise training using a running wheel or the control group. Subsequently, two groups were injected with aldosterone (0.125 μg kg-1 min-1) and administered 1% NaCl water, and two groups were administered aldosterone only in solvent 4 weeks after initiating the exercise. Aldosterone was injected for another 4 weeks, and NaCl water was administered from 5 weeks after starting the exercise until 8 weeks. Although both aldosterone and NaCl water significantly decreased the running distance, tubulointerstitial damage involving interstitial infiltration of macrophages and fibrosis and the elevation of urinary human L-FABP induced by aldosterone injection was prevented by voluntary running exercise training. Urinary human L-FABP levels were significantly correlated with the degree of tubulointerstitial damage. In conclusion, voluntary running exercise training delayed tubulointerstitial damage progression in the aldosterone-induced renal injury model and therefore may be a promising nonpharmacological strategy in CKD.

A ligand-induced structural change in fatty acid–binding protein 1 is associated with potentiation of peroxisome proliferator–activated receptor α agonists

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

Investigate the Existence of Domain Swapped Dimer in iLPB Family

Intracellular lipid binding proteins (iLBPs) are responsible for transport of different insoluble hydrophobic molecules in cytosol. Structures of members of this family include ten stranded beta barrel and two alpha helices which are like a cap for binding pocket of these proteins. Recently, we discovered a domain swapped dimer for Human cellular retinol binding protein II (hCRBPII) which is a member of iLBP. In Domain swapping two or more monomers exchange an identical part of their structures and form dimer or higher order oligomers. The swapped region in hCRBPII is three beta strands with two alpha helixes. Existence of domain swapping for this protein may have physiological relevant and may have huge effect on the folding pathway for iLBPs.1 We successfully expressed hCRPBII by Hela Cells and we got closer to find the size of hCRBPII in mammalian expression by using size exclusion chromatography and western blotting technique. The discovery of domain swapping in hCRBPII also led us to research domain swapping in other types of iLBPs, such as human fatty acid binding proteins. We studied domain swapping in fatty acid binding protein 5 (FABP5). FABP5 is responsible for endocannabinoid anandamide (AEA) transport in cytocol.2 We recently found the structure of domain swapped dimer for FABP5 binds to palmitic acid. Our eventual aim is to predict domain swapping from amino acid sequences in iLBPs.

Predicting Relative Protein Affinity of Novel Per- and Polyfluoroalkyl Substances (PFASs) by An Efficient Molecular Dynamics Approach.

With the phasing out of long-chain per- and polyfluoroalkyl substances (PFASs), production of a wide variety of alternative PFASs has increased to meet market demand. However, little is known about the bioaccumulation potential of these replacement compounds. Here, we developed a modeling workflow that combines molecular docking and molecular dynamics simulation techniques to estimate the relative binding affinity of a total of 15 legacy and replacement PFASs for human and rat liver-type fatty acid binding protein (hLFABP and rLFABP). The predicted results were compared with experimental data extracted from three different studies. There was good correlation between predicted free energies of binding and measured binding affinities, with correlation coefficients of 0.97, 0.79, and 0.96, respectively. With respect to replacement PFASs, our results suggest that EEA and ADONA are at least as strongly bound to rLFABP as perfluoroheptanoic acid (PFHpA), and as strongly bound to hLFABP as perfluorooctanoic acid (PFOA). For F-53 and F-53B, both have similar or stronger binding affinities than perfluorooctanesulfonate (PFOS). Given that interactions of PFASs with proteins (e.g., LFABPs) are important determinants of bioaccumulation potential in organisms, these alternatives could be as bioaccumulative as legacy PFASs, and are therefore not necessarily safer alternatives to long-chain PFASs.

Utility of urinary tubular markers for monitoring chronic tubulointerstitial injury after ischemia-reperfusion.

The aim of this study was to elucidate whether urinary tubular markers during the chronic phase of acute kidney injury (AKI) are associated with chronic tubulointerstitial damage. Male human L-type fatty acid binding protein (L-FABP) chromosomal transgenic (Tg) mice underwent ischaemic reperfusion (I/R) injury via renal pedicle clamping for either 10min or 20min. Contralateral nephrectomy was performed at the time of tissue reperfusion. The kidneys were analyzed 20days after the last I/R. Serum creatinine levels 20days post-I/R were significantly higher in the 20min I/R than in the 10min I/R and control groups and were similar between the 10min I/R and control groups. The degree of tubulointerstitial damage 20days post-I/R was significantly more severe in the 20min I/R than in the 10min I/R and control groups, as well as in the 10min I/R than in the control group. Urinary levels of human L-FABP, albumin, and kidney injury molecule-1 (KIM-1) 20days post-I/R were significantly higher in the 20min I/R than in the control group, whereas urinary L-FABP was significantly higher in the 10min I/R than in the control group. Conversely, urinary neutrophil gelatinase-associated lipocalin levels did not significantly differ between the three groups. Finally, the urinary levels of human L-FABP, albumin, and KIM-1 levels 20days post-I/R were significantly correlated with the degree of renal damage. Urinary levels of human L-FABP, albumin and, KIM-1 may be useful for monitoring AKI-to-CKD transition in clinical practice.

Investigating the Conformational Transitions of Human Adipocyte Fatty Acid Binding Protein Upon Binding Leukotriene B4 by Solution-State NMR Spectroscopy

Investigating the Conformational Transitions of Human Adipocyte Fatty Acid Binding Protein Upon Binding Leukotriene B4 by Solution-State NMR Spectroscopy

A quantum dot-based lateral flow immunoassay for the sensitive detection of human heart fatty acid binding protein (hFABP) in human serum

We describe the preparation and validation of a novel lateral flow immunoassay test for the detection of human heart fatty acid binding protein (hFABP). Water-soluble CdTe quantum dots (QDs) were selected as the fluorescent label and were linked covalently to anti-hFABP antibodies. Upon conjugation, the secondary structure of the anti-hFABP was preserved and the fluorescence quantum yield of the CdTe QDs increased. The labelled antibodies were transferred to the immunoassay test strip and the antigen–antibody reaction was successfully performed. This evidenced the preserved antibody activity of QD-labelled anti-hFABP towards hFABP, and provided a rapid means for the quantitation of hFABP in human serum within the range of 0–160ng ∙ ml−1, with a much lower detection limit of 221pg.∙ ml−1 compared with other rapid tests based on lateral flow immunoassays. This new immunoassay test has been successfully used for the early detection of acute myocardial infarction.

Endocannabinoid Interaction with Human FABP1: Impact of the T94A Variant

Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and a variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and the T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide and 2-arachidonoylglycerol 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 the T94A variant did not differ in affinity for AEA and most other NAEs, it exhibited a modestly higher affinity for OEA, as well as a higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1's secondary structure more extensively than any other previously examined ligand did. The T94A variant without a ligand was more susceptible to temperature-induced unfolding. While the T94A variant was much less sensitive to ligand (i.e., AEA or 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized the 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 strongly 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.

Cytotoxicity of novel fluorinated alternatives to long-chain perfluoroalkyl substances to human liver cell line and their binding capacity to human liver fatty acid binding protein.

Although shorter chain homologues and other types of fluorinated chemicals are currently used as alternatives to long-chain perfluoroalkyl substances (PFASs), their safety information remains unclear and urgently needed. Here, the cytotoxicity of several fluorinated alternatives (i.e., 6:2 fluorotelomer carboxylic acid (6:2 FTCA), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), and hexafluoropropylene oxide (HFPO) homologues) to human liver HL-7702 cell line were measured and compared with perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Their binding mode and affinity to human liver fatty acid binding protein (hL-FABP) were also determined. Compared with PFOA and PFOS, 6:2 Cl-PFESA, HFPO trimer acid (HFPO-TA), HFPO tetramer acid (HFPO-TeA), and 6:2 FTSA showed greater toxic effects on cell viabilities. At low exposure doses, these alternatives induced cell proliferation with similar mechanism which was different from that of PFOA and PFOS. Furthermore, binding affinity to hL-FABP decreased in the order of 6:2 FTCA<6:2 FTSA<HFPO dimer acid (HFPO-DA)<PFOA<PFOS/6:2 Cl-PFESA/HFPO-TA. Due to their distinctive structure, 6:2 Cl-PFESA and HFPO homologues were bound to the hL-FABP inner pocket with unique binding modes and higher binding energy compared with PFOA and PFOS. This research enhances our understanding of the toxicity of PFAS alternatives during usage and provides useful evidence for the development of new alternatives.

P-56 ヒト心臓由来脂肪酸結合タンパク質の脂肪酸認識機構研究(ポスター発表の部)

P-56 ヒト心臓由来脂肪酸結合タンパク質の脂肪酸認識機構研究(ポスター発表の部)

Molecular differences between human liver fatty acid binding protein and its T94A variant in their unbound and lipid-bound states

Liver fatty acid binding protein (L-FABP) is an abundant cytosolic protein playing a central role in intracellular lipid trafficking. The L-FABP T94A variant, originating from one of the most common polymorphisms in the FABP family, is associated with several lipid-related disorders. However, the molecular factors that determine the observed functional differences are currently unknown.In our work, we performed a high resolution comparative molecular analysis of L-FABP T94T and L-FABP T94A in their unbound states and in the presence of representative ligands of the fatty acid and bile acid classes. We collected residue-resolved NMR spectral fingerprints of the two variants, and compared secondary structures, backbone dynamics, side chain arrangements, binding site occupation, and intermolecular contacts. We found that threonine to alanine replacement did not result in strongly perturbed structural and dynamic features, although differences in oleic acid binding by the two variants were detected. Based on chemical shift perturbations at sites distant from position 94 and on differences in intermolecular contacts, we suggest that long-range communication networks in L-FABP propagate the effect of amino acid substitution at sites relevant for ligand binding or biomolecular recognition.