Estudo estrutural e funcional das proteínas ligadoras de ácidos graxos (FABP- Fatty Acid Binding Proteins) de Fasciola hepatica

As proteínas ligadoras de ácidos graxos (Fatty Acid Binding Proteins, FABPs) de parasitos têm um papel importante no processo de infecção por estes organismos. Por este motivo, estas proteínas são antígenos candidatos para vacina contra a infecção por Schistosoma mansoni e Fasciola hepatica. No presente trabalho foram caracterizadas FABPs de F. hepatica e comparadas com a proteína Sm14 de S. mansoni, a FABP de parasito melhor caracterizada, mediante análise de sequências e estruturas modeladas. Também foram clonadas, expressas e purificadas as FABPs tipo 1 e tipo 3 de F. hepatica. Os resultados do presente estudo indicam que a FABP tipo 3 de F. hepatica é relacionada estrutural, imunológica e funcionalmente com a Sm14, um candidato vacinal amplamente estudado. Devido à importância da Sm14 como alvo para o desenvolvimento de vacina para a esquistossomose, as características apresentadas pela FhFABP3 de F. hepatica apontam esta proteína como um candidato importante também para o desenvolvimento de uma vacina contra a fasciolose

Probing the interaction of brain fatty acid binding protein (B-FABP) with model membranes

Brain fatty acid-binding protein (B-FABP) interacts with biological membranes and delivers polyunsaturated fatty acids (FAs) via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that BFABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II) that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L-FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L-FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L-FABP mRNA level. Full-length L-FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA-FABP). Chang cells were stably transfected with pc-DNA-FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 mu mol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L-FABP was documented by regular reverse transcription polyrnerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot. The pcDNA-FABP-transfected cells expressed full-length L-FABP mRNA, which was absent from vector-transfected control cells. Western blot showed expression of 14-kd L-FABP protein in pcDNA-FABP-transfected cells, but not in vector-transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L-FABP expression. Lower LDH release was observed in the higher L-FABP-expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L-FABP gene. The L-FABP-expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L-FABP has a significant role in oxidative stress.

LPS-binding protein and IL-6 Mark paradoxical tuberculosis immune reconstitution inflammatory syndrome in HIV patients

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS is becoming increasingly apparent, however the potential involvement in TB-IRIS of a leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not been investigated before. Here we aimed to investigate the innate nature of the cytokine response in TB-IRIS and to identify novel potential biomarkers. Methods: From a large prospective cohort of HIV-TB co-infected patients receiving TB treatment, we compared 40 patients who developed TB-IRIS during the first month of ART with 40 patients matched for age, sex and baseline CD4 count who did not. We analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14, endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and anti-inflammatory cytokines before and during ART. Results: We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013) compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in multivariate models containing significant cytokines from pre-ART (p = 0.039) and during TB-IRIS (p = 0.034). Conclusion: We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP levels during IRIS-event as potential biomarkers in TB-IRIS. Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6 holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS. Future clinical studies should investigate the importance and clinical relevance of these markers for the diagnosis and treatment of TB-IRIS. Copyright: © 2013 Goovaerts et al.

Probabilistic classification of acute myocardial infarction from multiple cardiac markers

Logistic regression and Gaussian mixture model (GMM) classifiers have been trained to estimate the probability of acute myocardial infarction (AMI) in patients based upon the concentrations of a panel of cardiac markers. The panel consists of two new markers, fatty acid binding protein (FABP) and glycogen phosphorylase BB (GPBB), in addition to the traditional cardiac troponin I (cTnI), creatine kinase MB (CKMB) and myoglobin. The effect of using principal component analysis (PCA) and Fisher discriminant analysis (FDA) to preprocess the marker concentrations was also investigated. The need for classifiers to give an accurate estimate of the probability of AMI is argued and three categories of performance measure are described, namely discriminatory ability, sharpness, and reliability. Numerical performance measures for each category are given and applied. The optimum classifier, based solely upon the samples take on admission, was the logistic regression classifier using FDA preprocessing. This gave an accuracy of 0.85 (95% confidence interval: 0.78-0.91) and a normalised Brier score of 0.89. When samples at both admission and a further time, 1-6 h later, were included, the performance increased significantly, showing that logistic regression classifiers can indeed use the information from the five cardiac markers to accurately and reliably estimate the probability AMI. © Springer-Verlag London Limited 2008.

Protein expression profiling during chick retinal maturation: a proteomics-based approach

Background: The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E) development commencing at day 12 through 13, 17, 19 and post-hatch (P) 1 and 33 days.

Results: 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF), PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line), nucleophosmin 1 (NPM1), dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP), beta-synuclein and enhancer of rudimentary homologue.

Conclusion: This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

Novel biomarkers in early diagnosis of acute myocardial infarction compared with cardiac troponin T

Aims: To evaluate the role of novel biomarkers in early detection of acute myocardial infarction (MI) in patients admitted with acute chest pain.
Methods and results: A prospective study of 664 patients presenting to two coronary care units with chest pain was conducted over 3 years from 2003. Patients were assessed on admission: clinical characteristics, ECG (electrocardiogram), renal function, cardiac troponin T (cTnT), heart fatty acid binding protein (H-FABP), glycogen phosphorylase-BB, NT-pro-brain natriuretic peptide, D-dimer, hsCRP (high sensitivity C-reactive protein), myeloperoxidase, matrix metalloproteinase-9, pregnancy associated plasma protein-A, soluble CD40 ligand. A =12 h cTnT sample was also obtained. MI was defined as cTnT = 0.03 µg/L. In patients presenting <4 h of symptom onset, sensitivity of H-FABP for MI was significantly higher than admission cTnT (73 vs. 55%; P = 0.043). Specificity of H-FABP was 71%. None of the other biomarkers challenged cTnT. Combined use of H-FABP and cTnT (either one elevated initially) significantly improved the sensitivities of H-FABP or cTnT (85%; P = 0.004). This combined approach also improved the negative predictive value, negative likelihood ratio, and the risk ratio.
Conclusion: Assessment of H-FABP within the first 4 h of symptoms is superior to cTnT for detection of MI, and is a useful additional biomarker for patients with acute chest pain.

Identificação de indicadores moleculares de bem-estar durante o cultivo de dourada (Sparus aurata, Linnaeus 1758), utilizando técnicas de proteómica

Dissertação de Mestrado, Biologia Marinha, Especialização em Biotecnologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Étude de l’implication de la protéine F du virus de l’hépatite C dans le développement de pathologie hépatique chez deux lignées de poissons zébrés transgéniques

La protéine core du virus de l’hépatite C (VHC) serait responsable des principaux effets pathogènes du VHC, dont le développement de fibrose, stéatose, cirrhose et carcinome hépatocellulaire. Un cadre de lecture alternatif existe dans le gène de core, permettant la synthèse d’une autre protéine appelée ARFP (pour alternatate reading frame protein) ou protéine F (pour frameshift), dont le rôle reste encore mal compris. La présence de la protéine F lors de l’étude des fonctions biologiques de core ne pouvant être exclue, il est possible que certains rôles attribués à core reflètent en réalité l’activité de la protéine F. Afin de déterminer les fonctions biologiques de la protéine F dans les hépatocytes et son influence dans la pathogenèse associée au VHC, nous avons généré des lignées transgéniques de poissons zébrés (Danio rerio) dans lesquelles l’expression de deux versions de la protéine F (AF11opti et AUG26opti) a été ciblée au foie par l’utilisation du promoteur de la liver fatty acid binding protein (L-FABP). Le phénotype des poissons transgéniques de génération F2 a été analysé au niveau morphologique, histologique et microscopique afin de rechercher des signes de pathologie hépatique. Nos résultats ont démontré l’implication de la protéine F dans le développement de stéatose hépatique chez les deux lignées transgéniques, mais aucun signe de fibrose ou d’oncogenèse n’a été détecté. L’identification des mécanismes cellulaires et moléculaires responsables de l’accumulation lipidique induite par la protéine F pourrait permettre de mieux comprendre son rôle dans la pathogenèse du VHC, et mener au développement de nouvelles stratégies antivirales.

Intestin et défauts métaboliques dans la résistance à l'insuline

En lien avec l’augmentation constante de l’obésité, de plus en plus de personnes sont atteintes de résistance à l’insuline ou de diabète de type 2. Ce projet doctoral s’est surtout intéressé à l’une des conséquences majeures des pathologies cardiométaboliques, soit la dyslipidémie diabétique. À cet égard, les gens présentant une résistance à l’insuline ou un diabète de type 2 sont plus à risque de développer des perturbations lipidiques caractérisées essentiellement par des taux élevés de triglycérides et de LDL-cholestérol ainsi que de concentrations restreintes en HDL-cholestérol dans la circulation. Les risques de maladies cardiovasculaires sont ainsi plus élevés chez ces patients. Classiquement, trois organes sont connus pour développer l’insulino-résistance : le muscle, le tissu adipeux et le foie. Néanmoins, certaines évidences scientifiques commencent également à pointer du doigt l’intestin, un organe critique dans la régulation du métabolisme des lipides postprandiaux, et qui pourrait, conséquemment, avoir un impact important dans l’apparition de la dyslipidémie diabétique. De façon très intéressante, des peptides produits par l’intestin, notamment le GLP-1 (glucagon-like peptide-1), ont déjà démontré leur potentiel thérapeutique quant à l’amélioration du statut diabétique et leur rôle dans le métabolisme intestinal lipoprotéinique. Une autre évidence est apportée par la chirurgie bariatrique qui a un effet positif, durable et radical sur la perte pondérale, le contrôle métabolique et la réduction des comorbidités du diabète de type 2, suite à la dérivation bilio-intestinale. Les objectifs centraux du présent programme scientifique consistent donc à déterminer le rôle de l’intestin dans (i) l’homéostasie lipidique/lipoprotéinique en réponse à des concentrations élevées de glucose (à l’instar du diabète) et à des peptides gastro-intestinaux tels que le PYY (peptide YY); (ii) la coordination du métabolisme en disposant de l’AMPK (AMP-activated protein kinase) comme senseur incontournable permettant l’ajustement précis des besoins et disponibilités énergétiques cellulaires; et (iii) l’ajustement de sa capacité d’absorption des graisses alimentaires en fonction du gain ou de la perte de sa sensibilité à l’insuline démontrée dans les spécimens intestinaux humains prélevés durant la chirurgie bariatrique. Dans le but de confirmer le rôle de l’intestin dans la dyslipidémie diabétique, nous avons tout d’abord utilisé le modèle cellulaire intestinal Caco-2/15. Ces cellules ont permis de démontrer qu’en présence de hautes concentrations de glucose en basolatéral, telle qu’en condition diabétique, l’intestin absorbe davantage de cholestérol provenant de la lumière intestinale par l’intermédiaire du transporteur NPC1L1 (Niemann Pick C1-like 1). L’utilisation de l’ezetimibe, un inhibiteur du NPC1L1, a permis de contrecarrer cette augmentation de l’expression de NPC1L1 tout comme l’élévation de l’absorption du cholestérol, prouvant ainsi que le NPC1L1 est bel et bien responsable de cet effet. D’autre part, des travaux antérieurs avaient identifié certains indices quant à un rôle potentiel du peptide intestinal PYY au niveau du métabolisme des lipides intestinaux. Toutefois, aucune étude n’avait encore traité cet aspect systématiquement. Pour établir définitivement l’aptitude du PYY à moduler le transport et le métabolisme lipidique dans l’intestin, nous avons utilisé les cellules Caco-2/15. Notre étude a permis de constater que le PYY incubé du côté apical est capable de réduire significativement l’absorption du cholestérol et le transporteur NPC1L1. Puisque le rôle de l'AMPK dans l'intestin demeure inexploré, il est important non seulement de définir sa structure moléculaire, sa régulation et sa fonction dans le métabolisme des lipides, mais aussi d'examiner l'impact de l’insulino-résistance et du diabète de type 2 (DT2) sur son statut et son mode d’action gastro-intestinal. En employant les cellules Caco-2/15, nous avons été capables de montrer (i) la présence de toutes les sous-unités AMPK (α1/α2/β1/β2/γ1/γ2/γ3) avec une différence marquée dans leur abondance et une prédominance de l’AMPKα1 et la prévalence de l’hétérotrimère α1/β2/γ1; (ii) l’activation de l’AMPK par la metformine et l’AICAR, résultant ainsi en une phosphorylation accrue de l’enzyme acétylCoA carboxylase (ACC) et sans influence sur l'HMG-CoA réductase; (iii) la modulation négative de l’AMPK par le composé C et des concentrations de glucose élevées avec des répercussions sur la phosphorylation de l’ACC. D’autre part, l’administration de metformine au Psammomys obesus, un modèle animal de diabète et de syndrome métabolique, a conduit à (i) une régulation positive de l’AMPK intestinale (phosphorylation de l’AMPKα-Thr172); (ii) la réduction de l'activité ACC; (iii) l’augmentation de l’expression génique et protéique de CPT1, supportant une stimulation de la β-oxydation; (iv) une tendance à la hausse de la sensibilité à l'insuline représentée par l’induction de la phosphorylation d'Akt et l’inactivation de la phosphorylation de p38; et (v) l’abaissement de la formation des chylomicrons ce qui conduit à la diminution de la dyslipidémie diabétique. Ces données suggèrent que l'AMPK remplit des fonctions clés dans les processus métaboliques de l'intestin grêle. La preuve flagrante de l’implication de l’intestin dans les événements cardiométaboliques a été obtenue par l’examen des spécimens intestinaux obtenus de sujets obèses, suite à une chirurgie bariatrique. L’exploration intestinale nous a permis de constater chez ceux avec un indice HOMA élevé (marqueur d’insulinorésistance) (i) des défauts de signalisation de l'insuline comme en témoigne la phosphorylation réduite d'Akt et la phosphorylation élevée de p38 MAPK; (ii) la présence du stress oxydatif et de marqueurs de l'inflammation; (iii) la stimulation de la lipogenèse et de la production des lipoprotéines riches en triglycérides avec l’implication des protéines clés FABP, MTP et apo B-48. En conclusion, l'intestin grêle peut être classé comme un tissu insulino-sensible et répondant à plusieurs stimuli nutritionnels et hormonaux. Son dérèglement peut être déclenché par le stress oxydatif et l'inflammation, ce qui conduit à l'amplification de la lipogenèse et la synthèse des lipoprotéines, contribuant ainsi à la dyslipidémie athérogène chez les patients atteints du syndrome métabolique et de diabète de type 2.

Influence of birth weight on gene regulators of lipid metabolism and utilization in subcutaneous adipose tissue and skeletal muscle of neonatal pigs.

Epidemiological studies suggest that low-birth weight infants show poor neonatal growth and increased susceptibility to metabolic syndrome, in particular, obesity and diabetes. Adipose tissue development is regulated by many genes, including members of the peroxisome proliferator-activated receptor (PPAR) and the fatty acid-binding protein (FABP) families. The aim of this study was to determine the influence of birth weight on key adipose and skeletal muscle tissue regulating genes. Piglets from 11 litters were ranked according to birth weight and 3 from each litter assigned to small, normal, or large-birth weight groups. Tissue samples were collected on day 7 or 14. Plasma metabolite concentrations and the expression of PPARG2, PPARA, FABP3, and FABP4 genes were determined in subcutaneous adipose tissue and skeletal muscle. Adipocyte number and area were determined histologically. Expression of FABP3 and 4 was significantly reduced in small and large, compared with normal, piglets in adipose tissue on day 7 and in skeletal muscle on day 14. On day 7, PPARA and PPARG2 were significantly reduced in adipose tissue from small and large piglets. Adipose tissue from small piglets contained more adipocytes than normal or large piglets. Birth weight had no effect on adipose tissue and skeletal muscle lipid content. Low-birth weight is associated with tissue-specific and time-dependent effects on lipid-regulating genes as well as morphological changes in adipose tissue. It remains to be seen whether these developmental changes alter an individual's susceptibility to metabolic syndrome.

Exercise Training Reduces PGE(2) Levels and Induces Recovery from Steatosis in Tumor-bearing Rats

The effects of endurance training on PGE(2) levels and upon the maximal activity of hepatic carnitine palmitoyltransferase (CPT) system were studied in rats bearing the Walker 256 carciosarcoma. Animals were randomly assigned to a sedentary control (SC), sedentary tumor-bearing (ST), exercised control (EC), and as an exercised tumor-bearing (ET) group. Trained rats ran on a treadmill (60% VO(2) max) for 60 min/day, 5 days/week, for 8 weeks. We examined the mRNA expression (RT-PCR) and maximal activity (radio-assay) of the carnitine palmitoyltransferase system enzymes (CPT I and CPT II), as well as the gene expression of fatty-acid-binding protein (L-FABP) in the liver. PGE(2) content was measured in the serum, in tumor cells, and in the liver (ELISA). CPT I and CPT II maximal activity were decreased (p < 0.01) in ST when compared with SC. In contrast, serum PGE(2) was increased (p < 0.05) in cachectic animals as compared with SC. In the liver, PGE(2) content was also increased (p < 0.05) when compared with SC. Endurance training restored maximal CPT I and CPT II activity in the tumor-bearing animals (p < 0.0001). Exercise training induced PGE(2) levels to return to control values in the liver of tumor-bearing training rats (p < 0.05) and decreased the eicosanoid content in the tumor (p < 0.01). In conclusion, endurance training was capable of reestablishing liver carnitine palmitoyltransferase (CPT) system activity associated with decreased PGE(2) levels in cachectic tumor-bearing animals, preventing steatosis.

Characterization of the drug binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes), where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules. Thus, in addition to its central role in FA trafficking, I-FABP potentially serves as an important intracellular carrier of lipophilic drugs. In this study we provide a detailed thermodynamic analysis of the binding and stability properties of I-FABP in complex with a series of fibrate and fenamate drugs to provide an insight into the forces driving drug binding to I-FABP. Drug binding and selectivity for I-FABP are driven by the interplay of protein−ligand interactions and solvent processes. The Gibbs free energies (ΔG°) determined from dissociation constants at 25 °C ranged from −6.2 to −10 kcal/mol. The reaction energetics indicate that drug binding to I-FABP is an enthalpy−entropy driven process. The relationship between I-FABP stability and drug binding affinity was examined by pulse proteolysis. There is a strong coupling between drug binding and I-FABP stability. The effect of an I-FABP protein sink on the kinetics and thermodynamics of tolfenamic acid permeation across an artificial phospholipid membrane were investigated. I-FABP significantly decreased the energy barrier for desorption of tolfenamic acid from the membrane into the acceptor compartment. Taken together, these data suggest that the formation of stable drug−I-FABP complexes is thermodynamically viable under conditions simulating the reactant concentrations likely observed in vivo and maybe a significant biochemical process that serves as a driving force for passive intestinal absorption of lipophilic drugs.

Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.