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At least 19 recordsLinked to original sources
PubMed1993

Plasma protein binding of tacrolimus in humans.

An ultracentrifugation method was used for the determination of the tacrolimus (FK 506) free fraction in plasma. Samples of 400 microL were centrifuged for 6 h at 160,000 x g, and concentrations of [3H]tacrolimus were measured in the original plasma and in the second layer of the supernatant. The binding of tacrolimus in normal plasma averaged 73% and was independent of the drug concentration. Albumin (3.5%) binding averaged 44.4-57.0%, and alpha 1-acid glycoprotein (100 mg/L) binding averaged 39%. Hepatic transplant patients exhibited 72.0% +/- 3.6% binding of tacrolimus. Plasma protein binding is probably not an important factor in the disposition of tacrolimus.

Blood Proteins↗
PubMed2001

A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins.

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-Hsp90-FKBP52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport.

Active Transport, Cell Nucleus↗
PubMed2005

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506.

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.

Animals↗
PubMed1993

Probing hydration contributions to the thermodynamics of ligand binding by proteins. Enthalpy and heat capacity changes of tacrolimus and rapamycin binding to FK506 binding protein in D2O and H2O.

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atoms within the proteins and ligands and of these atoms with water. Delineation of the various energetic contributions of the stabilities of proteins or protein-ligand complexes in aqueous solution, and an evaluation of their structural basis, requires a direct account of the changes, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamycin, each bind with high affinity to a common site on a small FK506 binding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isomerase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the enthalpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solutions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titration calorimetry in the temperature range 5-30 degrees C. For both tacrolimus and rapamycin binding, a large enthalpic destabilization of binding is observed in D2O relative to H2O, in the temperature range examined. Additionally, large negative constant pressure heat capacity changes are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determinants of the differences in the energetics of binding in light and heavy water. The analysis suggests that a chief contributor to the observed enthalpic destabilization is the differential hydration, of protein and ligand atoms, by light and heavy water.

Carrier Proteins↗
PubMed2001

FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins.

The FK506 (tacrolimus)-binding protein (FKBP) type peptidyl-prolyl cis-trans isomerase (PPIase) in the hyperthermophilic archaeum Thermococcus sp. KS-1 was shown to be induced by temperature downshift to growth temperatures lower than the optimum. This PPIase (TcFKBP18) showed chaperone-like protein refolding activity in addition to PPIase activity in vitro. It refolded unfolded citrate synthase (CS) and increased the yield of the refolded protein. At a molar ratio of 15:1 ([TcFKBP18] to [CS]) in the refolding mixture, the recovered yield of folded CS was maximal at 62%, whereas that of spontaneous refolding was 11%. Increasing FKBP above a 15:1 ratio decreased the final yield, whereas the aggregation of unfolded CS was suppressed. A cross-linking analysis showed the formation of a complex between TcFKBP18 and unfolded CS (1:1 complex) at molar ratios of 3:1 to 15:1. However, molar ratios of 15:1 or 60:1 induced the binding of multiple FKBP molecules to an unfolded CS molecule (multimeric complex). Disrupting hydrophobic interaction by adding ethylene glycol at a molar ratio of 60:1 ([TcFKBP18] to [CS]) suppressed the formation of this multimeric complex, simultaneously enhancing CS refolding. FK506 also suppressed the formation of the multimeric complex while increasing the chaperone-like activity. These results suggest that the hydrophobic region of TcFKBP18, probably the FK506-binding pocket, was important for the interaction with unfolded proteins. No cross-linked product was detected between TcFKBP18 and native dimeric CS. TcFKBP18 probably traps the unfolded protein, then refolds and releases it in a native form. This FKBP might be important at growth temperatures lower than the optimum in Thermococcus sp. KS-1 cells.

Citrate (si)-Synthase↗
PubMed2001

Mutation of FKBP associated protein 48 (FAP48) at proline 219 disrupts the interaction with FKBP12 and FKBP52.

Immunophilins are known as intracellular receptors for the immunosuppressant drugs, cyclosporin A, FK506, and rapamycin. They can be divided into two groups, cyclophilins that bind cyclosporin A and FK506 binding proteins (FKBPs) that bind FK506 and rapamycin. Many efforts were made to elucidate the physiological role of the immunophilins. Many of them are involved in intracellular signalling as they bind to calcium channels or to steroid receptor complexes. A yeast two-hybrid screen was used to identify further target proteins that interact with known proteins. Recently, a 48-kDa FKBP associated protein (FAP48) was isolated that binds to FKBP12 and FKBP52. Binding of FAP48 to FKBPs is inhibited by the macrolide FK506 indicating that the binding sites on the immunophilins coincide with the binding site for FK506. A peptidyl-prolyl motif on FAP48 should be responsible for the binding of the protein to FKBPs. We sequentially point mutated proline sites on FAP48 and checked the mutant proteins for interaction with FKBP12 and FKBP52. Mutation of proline 219 to alanine leads to a loss of interaction indicating that a cysteinyl prolyl site might be responsible for the binding of FAP48 to FKBPs. Thus we identified proline 219 being essential for the interaction.

Adaptor Proteins, Signal Transducing↗
PubMed2001

2-Aryl-2,2-difluoroacetamide FKBP12 ligands: synthesis and X-ray structural studies.

[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.

Acetamides↗
PubMed2002

A kidney epithelial cell line from a Bolivian squirrel monkey.

Squirrel monkeys are the most commonly used New World primates in biomedical research, but in vitro studies are restricted by the limited number of cell lines available from this species. We report here the development and characterization of a continuous, kidney epithelial cell line (SQMK-FP cells) derived from a newborn squirrel monkey. Karyotype was consistent with Bolivian squirrel monkey (submetacentric chromosome pair 15 and acrocentric chromosome pair 16). All cells examined were hyperdiploid with chromosome numbers ranging from 52 to 57. Ultrastructural analysis of SQMK-FP cells revealed the presence of cell junctions with radiating filaments, indicating desmosomes and numerous surface projections containing longitudinally oriented filaments typical of tubular epithelium. Biochemically, SQMK-FP cells exhibit glucocorticoid resistance typical of the squirrel monkey. Glucocorticoid receptor (GR) binding is low in SQMK-FP cells because of high expression of the FK506-binding immunophilin FKBP51 that inhibits GR binding. SQMK-FP cells constitute a tubular epithelial cell line that has biochemical properties characteristic of squirrel monkeys and represents an alternate cell model to B-lymphoblast SML cells to study the biology of the squirrel monkey in vitro.

Animals↗
PubMed2005

Equilibrium studies of a fluorescent tacrolimus binding to surfactant protein A.

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent used in kidney, liver, and lung transplantation. The objective of this study was to characterize the binding of FK506 to surfactant protein A (SP-A), an abundant lipoprotein found in the alveolar fluid that functions as part of the innate immune system in the lung. We have synthesized a novel derivative of FK506 in which a dansyl moiety was covalently bound via cadaverine to the C22 position of the FK506 molecule (DNS-FK). Using the fluorescence and anisotropy properties of DNS-FK, we demonstrated that tacrolimus avidly binds to SP-A with an apparent equilibrium association constant (K(app)) of 10(7)M(-1) and a Gibbs binding free energy of -40 kJ mol(-1)K(-1). Derivatization of FK506 at the C22 position did not block FK506 binding to the cytosolic immunophilin FK506-binding protein (FK-BP) or human serum albumin (HSA), both used as controls of tacrolimus-binding proteins. K(app) for FK-BP/DNS-FK and HSA/DNS-FK complexes were 1.5 x 10(7) and 10(7)M(-1), respectively. The high sensitivity of this analytical technique makes it suitable for binding analysis of FK506 to proteins.

Fluorescence Polarization↗
PubMedDate not supplied

Validation of methods to study the distribution and protein binding of tacrolimus in human blood.

INTRODUCTION: Tacrolimus is a macrolide immunosuppressant that has a narrow therapeutic index, displays considerable variability in response, and has the potential for serious drug interactions. Therapeutic drug monitoring and dose individualisation for tacrolimus is complicated but essential. Few studies have investigated the blood distribution and protein binding of tacrolimus and the results of these studies are conflicting. The aim of the present study is to establish and validate methods to investigate the distribution of tacrolimus in human blood. To conduct these studies at clinically relevant concentrations the use of 3H-dihydro-tacrolimus instead of tacrolimus was investigated. METHODS: The use of radiolabelled tacrolimus was validated by conducting studies with a mixture of both labelled and unlabelled drug where tacrolimus was analysed by LC-MS/MS. The in vitro distribution of tacrolimus and 3H-dihydro-tacrolimus was investigated in blood collected from healthy subjects using Ficoll-Paque reagent and density gradient ultracentrifugation, respectively. The unbound fraction of tacrolimus in plasma was studied using equilibrium dialysis conducted at 37 degrees C. RESULTS: In blood, tacrolimus was found to be mainly associated with erythrocytes (85.3+/-1.5%), followed by diluted plasma proteins (14.3+/-1.5%) and lymphocytes (0.46+/-0.10%). In plasma, tacrolimus was found to mainly be associated with the soluble protein fraction (61.2+/-2.5%), high-density lipoproteins (HDL, 28.1+/-5.4%), low-density lipoproteins (LDL, 7.8+/-1.6%), and very low-density lipoproteins (VLDL, 1.4+/-0.3%). The unbound fraction of tacrolimus was found to be only 1.2+/-0.12%. Statistical comparison indicated that there was no significant difference in the blood distribution and plasma protein binding of 3H-dihydro-tacrolimus when compared with tacrolimus. DISCUSSION: These results have important implications for therapeutic drug monitoring of tacrolimus and subsequent studies of tacrolimus distribution in transplant recipients.

Chromatography↗
PubMed2006

A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Animals↗
PubMed2001

Microarrays of cells expressing defined cDNAs.

Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.

Animals↗
PubMed2006

Parallelized-over-parts computation of absolute binding free energy with docking and molecular dynamics.

We present a technique for biomolecular free energy calculations that exploits highly parallelized sampling to significantly reduce the time to results. The technique combines free energies for multiple, nonoverlapping configurational macrostates and is naturally suited to distributed computing. We describe a methodology that uses this technique with docking, molecular dynamics, and free energy perturbation to compute absolute free energies of binding quickly compared to previous methods. The method does not require a priori knowledge of the binding pose as long as the docking technique used can generate reasonable binding modes. We demonstrate the method on the protein FKBP12 and eight of its inhibitors.

Biophysics↗
PubMed1998

Differential expression and androgen regulation of the human selenium-binding protein gene hSP56 in prostate cancer cells.

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.

Androgens↗
PubMed2006

Analysis of calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue of heart failure by calstabin2 in mice.

The ryanodine receptor (RyR)/calcium-release channel on the sarcoplasmic reticulum mediates intracellular calcium release required for striated muscle contraction. RyR2, the predominant isoform in cardiac myocytes, comprises a macromolecular complex that includes calstabin2 (FKBP12.6). Calstabin2, an 11.8-kDa cis-trans peptidyl-prolyl isomerase (apparent molecular mass 12.6 kDa), stabilizes the closed state of the RyR2 channel, but the mechanism by which it achieves this regulation is not fully understood. Protein kinase A (PKA) phosphorylation of RyR2 decreases the affinity of calstabin2 for the RyR2 channel complex. In the present study we identified key aspartic acid residues on calstabin2 that are involved in binding to RyR2 and likely play a role in PKA phosphorylation-induced dissociation of calstabin2 from RyR2. We show that a mutant calstabin2 in which a key negatively charged residue (Asp-37) has been neutralized binds to a mutant RyR2 channel that mimics constitutively PKA-phosphorylated RyR2 (RyR2-S2808D). Furthermore, using wild-type and genetically altered murine models of heart failure induced by myocardial infarction, we show that manipulating the stoichiometry between calstabin2 and RyR2 can restore normal cardiac function in vivo.

Animals↗
PubMed2005

Structure-function analysis of squirrel monkey FK506-binding protein 51, a potent inhibitor of glucocorticoid receptor activity.

FK506-binding protein 51 (FKBP51) and FKBP52 are large molecular weight immunophilins that are part of the mature glucocorticoid receptor (GR) heterocomplex. These proteins possess peptidyl-prolyl isomerase (PPIase) and tetratricopeptide repeats (TPR) domains that are important for modulation of GR activity. A naturally occurring animal model of glucocorticoid resistance, the squirrel monkey, results from the relative overexpression of FKBP51 that renders the GR in a low-affinity state. In vitro studies demonstrated that the squirrel monkey form of FKBP51 is greater than 6-fold more potent than human FKBP51 in this respect. The goals of these studies were to determine the roles of the TPR and PPIase domains in the inhibitory activity of squirrel monkey FKBP51 and to gain insight into structural features of squirrel monkey FKBP51 responsible for potent inhibition of dexamethasone-stimulated GR activity. Mutations in the TPR of squirrel monkey FKBP51 that inhibit association with heat shock protein 90 blocked GR inhibitory activity. Mutations that abrogate the PPIase activity of squirrel monkey FKBP51 had no effect on GR inhibitory activity. Chimeras of squirrel monkey and human FKBP51 were tested to identify domains responsible for their different inhibitory potencies. Amino acid differences in domains FK1 and FK2 between squirrel monkey and human FKBP51 contribute equally to the enhanced inhibitory activity of squirrel monkey FKBP51. Furthermore, squirrel monkey FKBP51 in which either FK1 or FK2 was deleted lacked GR inhibitory activity. Thus, the potent inhibitory activity of squirrel monkey FKBP51 involves both FK domains and the heat shock protein 90-binding TPR domain.

Animals↗
PubMed2004

3D structure of human FK506-binding protein 52: implications for the assembly of the glucocorticoid receptor/Hsp90/immunophilin heterocomplex.

FK506-binding protein 52 (FKBP52), which binds FK506 and possesses peptidylprolyl isomerase activity, is an important immunophilin involved in the heterocomplex of steroid receptors with heat-shock protein 90. Here we report the crystal structures of two overlapped fragments [N(1-260) and C(145-459)] of FKBP52 and the complex with a C-terminal pentapeptide from heat-shock protein 90. Based on the structures of these two overlapped fragments, the complete putative structure of FKBP52 can be defined. The structure of FKBP52 is composed of two consecutive FKBP domains, a tetratricopeptide repeat domain and a short helical domain beyond the final tetratricopeptide repeat motif. Key structural differences between FKBP52 and FKBP51, including the relative orientations of the four domains and some important residue substitutions, could account for the differential functions of FKBPs.

Amino Acid Sequence↗