Human Ileal Bile Acid-binding Protein Research Articles

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling βD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Ligand entry in human ileal bile acid-binding protein is mediated by histidine protonation

Human ileal bile acid-binding protein (hI-BABP) has a key role in the intracellular transport of bile salts. To explore the role of histidine protonation in the binding process, the pH-dependence of bile salt binding and internal dynamics in hI-BABP was investigated using NMR spectroscopy and biophysical tools. Thermodynamic and kinetic measurements show an increase in the overall binding affinity and the association rate constant of the first binding step below the pKa of the histidines, suggesting that ligand binding is favoured by the protonated state. The overlap between residues exhibiting a high sensitivity to pH in their backbone amide chemical shifts and protein regions undergoing a global ms conformational exchange indicate a connection between the two processes. According to 15N NMR relaxation dispersion analysis, the slow motion is most pronounced at and above the pKa of the histidines. In agreement with the NMR measurements, MD simulations show a stabilization of the protein by histidine protonation. Hydrogen-bonding and van der Waals interactions mediating the flow of information between the C/D- and G/H-turn regions hosting the three histidines, suggest a complex way of pH-governed allosteric regulation of ligand entry involving a transition between a closed and a more open protein state.

Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein.

Human ileal bile acid-binding protein (I-BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid-binding proteins (iLBPs), disorder-order transitions and local unfolding processes are thought to mediate ligand entry and release in human I-BABP. To gain insight into the stability of various protein regions, the temperature response of human I-BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue-specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate β-strands contains a substantial amount of residual structure, whereas several segments of the C-terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the β-barrel, the E-F region and part of the helical cap. MD simulations up to 1μs show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D-E turn and E-F region in the initiation of unfolding. The response of human I-BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.

The long variant of human ileal bile acid-binding protein associated with colorectal cancer exhibits sub-cellular localization and lipid binding behaviour distinct from those of the common isoform

BackgroundIleal bile acid-binding protein, IBABP, participates in the intracellular trafficking of bile salts and influences their signaling activities. The recently discovered variant, IBABP-L, bearing an N-terminal 49-amino acid extension, was found to be associated with colorectal cancer and to protect cancer cells from the cytotoxic effects of deoxycholate. However, the precise function and the molecular properties of this variant are currently unknown. MethodsBioinformatics tools and confocal microscopy were used to investigate the sub-cellular localization of IBABP-L; protein dynamics, ligand binding and interaction with membrane models were studied by 2D NMR and fluorescence spectroscopy. ResultsBased on sub-cellular localization experiments we conclude that IBABP-L is targeted to the secretory pathway by a 24-residue signal peptide and, upon its cleavage, the mature protein is constitutively released into the extracellular space. Site-resolved NMR experiments indicated the distinct preference of primary and secondary bile salts to form either heterotypic or homotypic complexes with IBABP-L. The presence of the relatively dynamic N-terminal extension, originating only subtle conformational perturbations in the globular domain, was found to influence binding site occupation in IBABP-L as compared to IBABP. Even more pronounced differences were found in the tendency of the two variants to associate with phospholipid bilayers. ConclusionsIBABP-L exhibits different sub-cellular localization, ligand-binding properties and membrane interaction propensity compared to the canonical short isoform. General significanceOur results constitute an essential first step towards an understanding of the role of IBABP-L in bile salt trafficking and signaling under healthy and pathological conditions.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR.

The coordinates of the 10 lowest energy structures of the human I-BABP : GCDA : GCA complex as well as the distance restraints used to calculate the final ensemble have been deposited in the Brookhaven Protein Data Bank with accession number 2MM3.

Temperature dependence of backbone dynamics in human ileal bile acid-binding protein: implications for the mechanism of ligand binding.

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the apo form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond-nanosecond (ps-ns) backbone flexibility. In the current study, temperature-dependent (15)N NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the apo state can be grouped into two clusters with slightly different exchange rates. The entropy-enthalpy compensation observed for both clusters suggests a disorder-order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps-ns motion of (15)N-(1)H bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the apo and holo states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein.

Kinetic Mechanism of Ligand Binding in Human Ileal Bile Acid Binding Protein as Determined by Stopped-Flow Fluorescence Analysis

Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

Determinants of Cooperativity and Site Selectivity in Human Ileal Bile Acid Binding Protein

Human ileal bile acid binding protein (I-BABP) is a member of the family of intracellular lipid-binding proteins and is thought to play a role in the enterohepatic circulation of bile salts. Our group has previously shown that human I-BABP binds two molecules of glycocholate (GCA) with low intrinsic affinity but an extraordinary high degree of positive cooperativity. Besides the strong positive cooperativity, human I-BABP exhibits a high degree of site selectivity in its interactions with GCA and glycochenodeoxycholate (GCDA), the two major bile salts in humans. In this study, on the basis of our first generation nuclear magnetic resonance (NMR) structure of the ternary complex of human I-BABP with GCA and GCDA, we introduced single-residue mutations at certain key positions in the binding pocket that might disrupt a hydrogen-bonding network, a likely way of energetic communication between the two sites. Macroscopic binding parameters were determined using isothermal titration calorimetry, and site selectivity was monitored by NMR spectroscopy of isotopically enriched bile salts. According to our results, cooperativity and site selectivity are not linked in human I-BABP. While cooperativity is governed by a subtle interplay of entropic and enthalpic contributions, site selectivity appears to be determined by more localized enthalpic effects. Possible communication pathways between the two binding sites are discussed.

Ileal Bile Acid-binding Protein, Functionally Associated with the Farnesoid X Receptor or the Ileal Bile Acid Transporter, Regulates Bile Acid Activity in the Small Intestine

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

The gene encoding the human ileal bile acid-binding protein (I-BABP) is regulated by peroxisome proliferator-activated receptors

Peroxisome proliferator-activator receptors (PPAR) are involved in cholesterol homeostasis through the regulation of bile acids synthesis, composition, and reclamation. As ileal bile acid-binding protein (I-BABP) is thought to play a crucial role in the enterohepatic circulation of bile acids, we investigated whether I-BABP gene expression could also be affected by PPAR. Indeed, treatment with the PPARα-PPARβ/δ agonist bezafibrate led to the up-regulation of I-BABP mRNA levels in the human intestine-derived Caco-2 cells. Cotransfections of the reporter-linked human I-BABP promoter (hI-BABP −2769/+44) together with PPAR and RXR expression vectors demonstrated that the fibrate-mediated induction of the I-BABP gene is dependent on PPARα or PPARβ/δ. Using progressive 5′ deletions of the hI-BABP promoter and sequence analysis, we identified a putative PPAR-binding site located at the position −198 and −186 upstream of the transcription initiation site. Electrophoretic mobility shift assays showed that the PPAR/RXR heterodimer can specifically bind to this PPRE-like motif. The deletion of the PPRE within the hI-BABP promoter abolished the PPAR-mediated transactivation in transient transfection assays. The regulation of the I-BABP promoter by PPAR appears species-specific, as the mouse I-BABP promoter, which lacks a conserved PPRE, was not responsive to exogenous PPAR expression in the presence of bezafibrate. Our findings show that the I-BABP gene may be a novel target for PPAR in humans and further emphasize the role for PPAR in the control of bile acid homeostasis.

Human ileal bile acid-binding protein promoter and the effects of CDX2

The ileal bile acid-binding protein (IBABP) is important for the reabsorption of bile salts in the distal small intestine. Studies with the human IBABP gene ( FABP6, on chromosome 5q33.3–q34) defined the major transcription start site and identified conserved elements. A consensus element for the caudal-related homeobox factor CDX2 was functional in gel-shift assays and in transfection experiments.

FXRE can function as an LXRE in the promoter of human ileal bile acid-binding protein (I-BABP) gene

Ileal bile acid-binding protein (I-BABP) is a 14 kDa cytosolic protein which binds bile acids with a high affinity. It is thought to be implicated in the enterohepatic circulation of bile acids and, hence, in cholesterol homeostasis. Using a combination of in vivo and in vitro experiments, we have recently shown that I-BABP gene expression can be indirectly up-regulated by cholesterol through the activation of sterol-responsive element-binding protein 1c (SREBP1c) by liver X-receptor (LXR). We report here that I-BABP can be also a direct target for LXR. I-BABP regulation by LXR is maintained when the SREBP binding site is deleted in the I-BABP promoter and occurs, in the absence of conventional LXRE sequences, through an IR1 sequence previously identified as a farnesoid X-receptor-responsive element (FXRE). Electrophoretic mobility shift assays demonstrated that the LXR/RXR heterodimer specifically recognizes the FXRE. Collectively, these data strongly suggest that LXR can regulate the I-BABP gene by both direct and indirect mechanisms.

Steroid ring hydroxylation patterns govern cooperativity in human bile acid binding protein.

Human ileal bile acid binding protein (I-BABP) is a member of the intracellular lipid binding protein family. This protein is thought to function in the transcellular transport and enterohepatic circulation of bile salts. Human I-BABP binds two molecules of glycocholate, the physiologically most abundant bile salt, with modest intrinsic affinity but a remarkably high degree of positive cooperativity. Here we report a calorimetric analysis for the binding of a broad panel of bile salts to human I-BABP. The interaction of I-BABP with nine physiologically relevant derivatives of cholic acid, chenodeoxycholic acid, and deoxycholic acid in their conjugated (glycine and taurine) and unconjugated forms was monitored by isothermal titration calorimetry. All bile salts bound to I-BABP with a 2:1 stoichiometry and similar overall affinity, but the derivatives of cholic acid displayed much higher Hill coefficients, a measure of macroscopic positive cooperativity. To test whether the cooperativity was dependent on individual structural features of the bile salt side chain, a series of side-chain-extended bile salts that lacked a hydrogen bond donor or acceptor at C-24 were chemically synthesized. These synthetic variants exhibited the same energetic and cooperativity profile as the naturally occurring bile salts. Our findings indicate that cooperativity in bile salt-I-BABP recognition is governed by the pattern of steroid B- and C-ring hydroxylation and not the presence or type of side-chain conjugation.

Sterol Regulatory Element-binding Protein-1c Is Responsible for Cholesterol Regulation of Ileal Bile Acid-binding Protein Gene in Vivo: POSSIBLE INVOLVEMENT OF LIVER-X-RECEPTOR

Ileal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acid (BA) specifically. In the ileum, it is thought to be implied in their enterohepatic circulation. Because the fecal excretion of BA represents the main physiological way of elimination for cholesterol (CS), the I-BABP gene could have a major function in CS homeostasis. Therefore, the I-BABP gene expression might be controlled by CS. I-BABP mRNA levels were significatively increased when the human enterocyte-like CaCo-2 cells were CS-deprived and repressed when CS were added to the medium. A highly conserved sterol regularory element-like sequence (SRE) and a putative GC box were found in human I-BABP gene promoter. Different constructs of human I-BABP promoter, cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene, have been used in transfections studies. CAT activity of the wild type promoter was increased in presence of CS-deprived medium, and conversely, decreased by a CS-supplemented medium. The inductive effect of CS depletion was fully abolished when the putative SRE sequence and/or GC box were mutated or deleted. Co-transfections experiments with the mature isoforms of human sterol responsive element-binding proteins (SREBPs) and Sp1 demonstrate that the CS-mediated regulation of I-BABP gene was dependent of these transcriptional factors. Paradoxically, mice subjected to a standard chow supplemented with 2% CS for 14 days exhibited a significant rise in both I-BABP and SREBP1c mRNA levels. We show that in vivo, this up-regulation could be explained by a recently described regulatory pathway involving a positive regulation of SREBP1c by liver-X-receptor following a high CS diet.

P-175 Hepatic injury and regeneration after transient ischemia of rat liver

Peroxisome proliferator-activator receptors (PPAR) are involved in cholesterol homeostasis through the regulation of bile acids synthesis, composition, and reclamation. As ileal bile acid-binding protein (I-BABP) is thought to play a crucial role in the enterohepatic circulation of bile acids, we investigated whether I-BABP gene expression could also be affected by PPAR. Indeed, treatment with the PPARα-PPARβ/δ agonist bezafibrate led to the up-regulation of I-BABP mRNA levels in the human intestine-derived Caco-2 cells. Cotransfections of the reporter-linked human I-BABP promoter (hI-BABP−2769/+44) together with PPAR and RXR expression vectors demonstrated that the fibrate-mediated induction of the I-BABP gene is dependent on PPARα or PPARβ/δ. Using progressive 5′ deletions of the hI-BABP promoter and sequence analysis, we identified a putative PPAR-binding site located at the position −198 and −186 upstream of the transcription initiation site. Electrophoretic mobility shift assays showed that the PPAR/RXR heterodimer can specifically bind to this PPRE-like motif. The deletion of the PPRE within the hI-BABP promoter abolished the PPAR-mediated transactivation in transient transfection assays. The regulation of the I-BABP promoter by PPAR appears species-specific, as the mouse I-BABP promoter, which lacks a conserved PPRE, was not responsive to exogenous PPAR expression in the presence of bezafibrate. Our findings show that the I-BABP gene may be a novel target for PPAR in humans and further emphasize the role for PPAR in the control of bile acid homeostasis.