BIOINFORMATICS, TISSUE DISTRIBUTION, AND SUBCELLULAR LOCALIZATION ANALYSES OF FK506 BINDING PROTEIN 12B FROM SILKWORMS.

FK506 binding proteins (FKBPs) are intracellular receptors of the immunosuppressant FK506 and play important roles in the correct folding of new proteins and the self-assembly of biological macromolecules. FKBP12 is a member of the FKBP family that is widely expressed and highly conserved in many species. In this study, we identified the complete cDNA sequence encoding the FKBP12 ortholog in Bombyx mori, named Bm-FKBP12B (GenBank accession no. DQ443423). Multiple-sequence alignment among different species revealed a high similarity among FKBP12 paralogs and orthologs. Bioinformatics analysis of the Bm-FKBP12B gene showed that it is located on chromosome 20 and consists of three exons and two introns. We cloned, expressed, and purified the Bm-FKBP12B protein in Escherichia coli and generated a specific polyclonal antibody against Bm-FKBP12B. The real-time quantitative reverse-transcription (qRT) PCR and Western blotting results showed that Bm-FKBP12B was present throughout all of the development stages, but it was abundant in the adult and embryo stages. Bm-FKBP12B expression was higher in the silk gland and gut, suggesting that it might play important roles in regulating gene expression in the silk gland and during silk fiber formation. Bm-FKBP12B protein was distributed in the cytoplasm, nucleus, and nuclear membrane.

Nocardiopsins: new FKBP12-binding macrolide polyketides from an Australian marine-derived actinomycete, Nocardiopsis sp.

A marine-derived actinomycete, Nocardiopsis sp. (CMB-M0232), obtained from a sediment sample collected at a depth of 55 m off the coast of Brisbane, Australia, yielded two new macrolide polyketides. Structures for nocardiopsins A and B were assigned by detailed spectroscopic analysis, degradation and chemical derivatization. A Marfey's analysis revealed an unexpected acid-mediated partial racemization of the L-pipecolic acid incorporated within the nocardiopsins. The scope of this racemization was assessed against a selection of natural and synthetic N-acyl pipecolic acids. While the nocardiopsins are not antibacterial, antifungal or cytotoxic, they do exhibit low-micromolar binding to the immunophilin FKBP12, consistent with their structural and biosynthetic relationship to the immunosuppressive agents FK506 and rapamycin. The nocardiopsins represent a new point of entry into what has been a valuable, exclusive and reclusive region of bioactive chemical space--that surrounding the FK506/rapamycin pharmacophore.

Selective elution of target protein from affinity resins by a simple reductant with a thiol group.

We have made a chance discovery of selective elution of a specific binding protein from affinity resins by mixing them with aqueous solutions of a widely used reductant, 2-mercaptoethanol (2ME), under mild conditions. Our studies suggest this phenomenon would be generic, and could be a powerful method for identification of a specific binding protein. We here exhibit the experimental conditions and successful examples in which target proteins of benzensulfonamide and FK506 were selectively eluted from affinity resins bearing these compounds, while non-specific ones remained.

Identification of small molecule binding molecules by affinity purification using a specific ligand immobilized on PEGA resin.

We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.

Drug receptor identification from multiple tissues using cellular-derived mRNA display libraries.

The use of display technologies to identify small molecule receptors from proteome libraries would provide a significant advantage in drug discovery. We have used mRNA display to select, based on affinity, proteins that bind to a drug of interest. A library of mRNA-protein fusion molecules was constructed from human liver, kidney, and bone marrow transcripts and selected using an immobilized FK506-biotin conjugate. Three rounds of selection produced full-length FKBP12 (FK506 binding protein 12 kDa) as the dominant clone. An analogous method was also used to map the minimal drug binding domain within FKBP12. Using this approach, it is anticipated that mRNA display could eventually play a key role in the discovery and characterization of new drug receptor interactions.

High-throughput fluorescence polarization method for identification of FKBP12 ligands.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

Crystallization and preliminary X-ray diffraction analysis of FKBP12 complexed with a novel neurotrophic ligand.

A novel neurotrophic ligand, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester, has been complexed with FKBP12 and crystallized using the hanging-drop vapor-diffusion method. Crystals belong to P2(1) space group, with unit cell parameters a=41.2, b=29.6, c=41.5 A, beta=114.0 degrees. The crystals diffract to 1.8 A resolution limit.

[Identification rhFKBP12 by ESI-quadrupole-oa-TOF tandem mass spectrometry].

AIM: To identify recombinant protein rhFKBP12 by new technique ESI-quadrupole-oa-TOF tandem mass spectrometry. METHODS: The molecular weight of rhFKBP12 was measured by ESI-MS. Digest rhFKBP12 by trypsin at 37 degrees C over night. The tryptic digested peptides were measured and then two doublely charged peptides were selected to measure their amino acid sequence by ESI-MS/MS. Search database with the measured amino acid sequence to identify rhFKBP12. RESULTS: The calculated molecular weight of rhFKBP12 was 11,819.54 and the measured value was 11,820.38. The measurement percent error was only 0.007%. The sequence measured by ESI-MS/MS was QVETMS and EEGVAQMSV and then the database search results with them were both hFKBP12. CONCLUSION: The study proves that the primary structure of rhFKBP12 is correct and there is no amino acid deletion, mutation and modification in its expression, refolding and purification. It also shows that ESI-MS/MS is a good method to identify protein with advantage of sensitivity, high speed and accuracy.

A versatile method of identifying specific binding proteins on affinity resins.

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. Although solid-phase elution and competition methods conventionally are used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches often are severely restricted by low ligand solubility and/or slow kinetic dissociation. This article describes an alternative and versatile method, termed serial affinity chromatography, to identify ligand receptors using affinity resins.

Isolating the whole complex of target proteins of FK506 using affinity resins from novel solid phases.

The development of novel solid phases enabled us to create affinity resins that could be used to isolate the whole complex of target proteins responsible for the immunosuppressive effects of FK506 from rat brain lysate, whereas the affinity resins from commercially available matrices could not achieve this isolation. The results illustrate the enhanced effectiveness of the affinity resin made from this novel material at identifying the target protein of the bioactive compound compared to resins made from the well-known materials Affigel or Toyopearl. This effectiveness arises because the novel material is hydrophilic enough to reduce nonspecific binding proteins and because it has a higher density of ligands that capture the nonubiquitous target protein.

Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system.

The yeast two-hybrid assay has proven to be a powerful method to detect protein-protein interactions as well as to derive genome-wide protein interaction maps. More recently, three-hybrid assays have emerged as a means to detect both protein-RNA and protein-small molecule interactions. Despite the routine use of the two-hybrid assay and the potential of three-hybrid systems, there has been little quantitative characterization to understand how the strength of the protein interaction correlates with transcription activation. It is not known if the additional interaction in three-hybrid systems compromises the sensitivity of the system. Thus, here, we set out to determine the K(D) cutoff of a small molecule three-hybrid system and to determine if there is a correlation between the K(D) and the levels of transcription activation. A series of mutations to FK506-binding protein 12 (FKBP12) were designed to vary the affinity of this protein for the small molecule synthetic ligand for FK506-binding protein 12 (SLF). These FKBP12 variants were overexpressed and purified, and their K(D)'s for SLF were measured using a fluorescence polarization assay. Then the levels of transcription activation in a Mtx-DHFR yeast three-hybrid system were determined for these variants using a lacZ reporter gene. The K(D) cutoff of the Mtx yeast three-hybrid system is found to be ca. 50 nM. Further, the levels of transcription activation correlate with the strength of the binding interaction, though the dynamic range is only 1 order of magnitude. These results establish that the three-hybrid assay has the requisite sensitivity for drug discovery. However, the small dynamic range highlights a limitation to equilibrium-based assays for discriminating interactions based on affinity.