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1.
Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity. The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids. Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences. The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12. The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM). PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506. These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin. 相似文献
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3.
Ajit Prakash Sreekanth Rajan Ho Sup Yoon 《Protein science : a publication of the Protein Society》2016,25(4):905-910
Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506. 相似文献
4.
Mutation of FKBP associated protein 48 (FAP48) at proline 219 disrupts the interaction with FKBP12 and FKBP52 总被引:2,自引:0,他引:2
Neye H 《Regulatory peptides》2001,97(2-3):147-152
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. 相似文献
5.
The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein. 相似文献
6.
FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition. 总被引:6,自引:1,他引:6
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G Baughman G J Wiederrecht N F Campbell M M Martin S Bourgeois 《Molecular and cellular biology》1995,15(8):4395-4402
The immunosuppressive drugs FK506 and cyclosporin A block T-lymphocyte proliferation by inhibiting calcineurin, a critical signaling molecule for activation. Multiple intracellular receptors (immunophilins) for these drugs that specifically bind either FK506 and rapamycin (FK506-binding proteins [FKBPs]) or cyclosporin A (cyclophilins) have been identified. We report the cloning and characterization of a new 51-kDa member of the FKBP family from murine T cells. The novel immunophilin, FKBP51, is distinct from the previously isolated and sequenced 52-kDa murine FKBP, demonstrating 53% identity overall. Importantly, Western blot (immunoblot) analysis showed that unlike all other FKBPs characterized to date, FKBP51 expression was largely restricted to T cells. Drug binding to recombinant FKBP51 was demonstrated by inhibition of peptidyl prolyl isomerase activity. As judged from peptidyl prolyl isomerase activity, FKBP51 had a slightly higher affinity for rapamycin than for FK520, an FK506 analog. FKBP51, when complexed with FK520, was capable of inhibiting calcineurin phosphatase activity in an in vitro assay system. Inhibition of calcineurin phosphatase activity has been implicated both in the mechanism of immunosuppression and in the observed toxic side effects of FK506 in nonlymphoid cells. Identification of a new FKBP that can mediate calcineurin inhibition and is restricted in its expression to T cells suggests that new immunosuppressive drugs may be identified that, by virtue of their specific interaction with FKBP51, would be targeted in their site of action. 相似文献
7.
FK-506-binding proteins from streptomycetes producing immunosuppressive macrolactones of the FK-506 type. 总被引:4,自引:0,他引:4
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FK-506-binding proteins (FKBPs), which in T cells are supposed to mediate the immunosuppressive effects of the compounds FK-506 and rapamycin, have been isolated from Streptomyces chrysomallus, S. hygroscopicus subsp. ascomyceticus, and S. hygroscopicus. The latter two strains are producers of ascomycin (the ethyl analog of FK-506) and rapamycin, respectively. Like the 12-kDa FKBP in eukaryotic organisms such as humans, bovines, and Saccharomyces cerevisiae, or the FKBPs from gram-positive streptomycetes are peptidyl-prolyl-cis-trans isomerases. Inhibition studies using FK-506, rapamycin, or ascomycin, revealed inhibition of the peptidyl-prolyl cis-trans isomerase activity of the proteins at the nanomolar level, which is in the same range as with eukaryotic FKBPs. The M(r)s of the various FKBPs were 13,500 to 15,000, and they had the same pI of approximately 4.5. The N-terminal sequences of the three FKBPs were nearly identical in the first 20 amino acids. The amino acid sequence deduced from the gene sequence of S. chrysomallus gave a polypeptide of 124 amino acids. The homologies to FKBPs from humans, S. cerevisiae, and Neurospora crassa were 38, 39, and 50% identity in relevant positions, respectively. Significant homology of 38% was also seen with the C-terminal halves of bacterial protein surface antigens like the Mip protein of Legionella pneumophila and the 27-kDa Mip-like protein of Chlamydia trachomatis. In addition, two more open reading frames in Pseudomonas aeruginosa and Neisseria meningitidis of unknown function show regions of homology to the S. chrysomallus FKBP. In contrast to fungi, streptomycetes are resistant to macrolactones. Ascomycin-producing S. hygroscopicus subsp. ascomyceticus excretes the compound almost quantitatively into medium, which indicates that the organism has an efficient self-protection mechanism against its own secondary metabolite. 相似文献
8.
FK 506-binding proteins (FKBPs) are a family of cytosolic proteins identified by virtue of their ability to bind the immunosuppressants FK 506 and rapamycin. While their function has been extensively studied in the immune system, little is known about their role during early embryonic development. Here we describe the cloning and expression of a new Xenopus FKBP (xFKBP). xFKBP encodes a 63-kDa protein that shares high sequence homology with mouse FKBP65. It is expressed maternally and becomes restricted after the gastrula stage to dorsal mesoderm and notochord. At the tailbud stage expression persists in the notochord and begins to accumulate in epidermis, branchial arches and developing somites. In adults, xFKBP mRNA is confined to the testis. 相似文献
9.
The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae. 总被引:4,自引:2,他引:2
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J Heitman A Koller J Kunz R Henriquez A Schmidt N R Movva M N Hall 《Molecular and cellular biology》1993,13(8):5010-5019
The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway. 相似文献
10.
Immunophilins are protein chaperones with peptidylprolyl isomerase activity that belong to one of two large families, the cyclosporin-binding cyclophilins (CyPs) and the FK506-binding proteins (FKBPs). Each family displays characteristic and conserved sequence features that differ between the two families. We report a novel group of dual-family immunophilins that contain both CyP and FKBP domains for which we propose the name FCBP (FK506- and cyclosporin-binding protein). The FCBP of Toxoplasma gondii, a protozoan parasite, contained N-terminal FKBP and C-terminal CyP domains joined by tetratricopeptide repeats. Structure-function analysis revealed that both domains were functional and exhibited family-specific drug sensitivity. The individual domains of FCBP inhibited calcineurin (protein phosphatase 2B) in the presence of the appropriate drugs. In binding studies, FCBP recruited calcineurin in the presence of FK506 and a putative target of rapamycin homolog in the presence of rapamycin. Two additional FCBP sequences in Flavobacterium and one in Treponema (spirochete) were also identified in which the CyP and FKBP domains were in the reverse order. T. gondii growth was inhibited by cyclosporin and FK506 in a moderately synergistic manner. The knockdown of FCBP by RNA interference revealed its essentiality for T. gondii growth. Clearly, the FCBPs are novel chaperones and potential targets of multiple immunosuppressant drugs. 相似文献
11.
Weiwad M Edlich F Kilka S Erdmann F Jarczowski F Dorn M Moutty MC Fischer G 《Biochemistry》2006,45(51):15776-15784
Multiple intracellular receptors of the FK506 binding protein (FKBP) family of peptidylprolyl cis/trans-isomerases are potential targets for the immunosuppressive drug FK506. Inhibition of the protein phosphatase calcineurin (CaN), which has been implicated in the FK506-mediated blockade of T cell proliferation, was shown to involve a gain of function in the FKBP12/FK506 complex. We studied the potential of six human FKBPs to contribute to CaN inhibition by comparative examination of inhibition constants of the respective FK506/FKBP complexes. Interestingly, these FKBPs form tight complexes with FK506, exhibiting comparable dissociation constants, but the resulting FK506/FKBP complexes differ greatly in their affinity for CaN, with IC50 values in the range of 0.047-17 microM. The different capacities of FK506/FKBP complexes to affect CaN activity are partially caused by substitutions corresponding to the amino acid side chains K34 and I90 of FKBP12. Only the FK506 complexes of FKBP12, FKBP12.6, and FKBP51 showed high affinity to CaN; small interfering RNA against these FKBP allowed defining the contribution of individual FKBP in an NFAT reporter gene assay. Our results allow quantitative correlation between FK506-mediated CaN effects and the abundance of the different FKBPs in the cell. 相似文献
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Stuart L. Rulten Ross A. Kinloch Hilda Tateossian Colin Robinson Lucy Gettins John E. Kay 《Mammalian genome》2006,17(4):322-331
Analysis of the human repertoire of the FK506-binding protein (FKBP) family of peptidyl-prolyl cis/trans isomerases has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences
in tissue distribution and expression levels of each variant. In this article we describe the characterization of human FKBP19
(Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence
comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in
the acinar cells of mouse pancreas. 相似文献
14.
We report a novel chromatin-modulating factor, nuclear FK506-binding protein (FKBP). It is a member of the peptidyl prolyl cis-trans isomerase (PPIase) family, whose members were originally identified as enzymes that assist in the proper folding of polypeptides. The endogenous FKBP gene is required for the in vivo silencing of gene expression at the rDNA locus and FKBP has histone chaperone activity in vitro. Both of these properties depend on the N-terminal non-PPIase domain of the protein. The C-terminal PPIase domain is not essential for the histone chaperone activity in vitro, but it regulates rDNA silencing in vivo. Chromatin immunoprecipitation showed that nuclear FKBP associates with chromatin at rDNA loci in vivo. These in vivo and in vitro findings in nuclear FKBPs reveal a hitherto unsuspected link between PPIases and the alteration of chromatin structure. 相似文献
15.
The receptors for the immunosuppression drugs FK506 and rapamycin are called FKBPs (FK506-binding proteins). FKBPs comprise a large family; they are found in many species, including bacteria, fungi, animals, and plants. As a class of peptidyl-prolyl cis-trans isomerase enzymes, the FKBP genes have been the focus of recent studies on plant stress tolerance and immunology. We identified and analyzed gene families encoding these proteins in maize using computational and molecular biology approaches. Thirty genes were found to encode putative FKBPs according to their FK506-binding domain. The FKBP genes can be classified into single domain and multiple domain members based on the number of the domains. By analysis of the physical locations, the 30 FKBP genes were found to be widely distributed on 10 chromosomes. After analysis of the FKBP phylogenetic tree in the maize genome, we found that the 30 genes revealed two major clades. Gene duplication played a major role in the evolution of FKBP genes, which suggests that the FKBP genes in maize have a pattern significantly different from that of these genes in rice. Based on semi-quantitative RT-PCR, we found that the 30 FKBPs were expressed differently in various tissues in maize, which suggests that FKBP genes play different roles in each tissue. Several FKBPs were expressed at higher levels in roots, indicating that these genes in maize may have similar or overlapping functions. 相似文献
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A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus
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《The Journal of cell biology》1994,127(3):623-639
The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans- isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full- length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus. 相似文献
18.
Charged surface residues of FKBP12 participate in formation of the FKBP12-FK506-calcineurin complex.
R A Aldape O Futer M T DeCenzo B P Jarrett M A Murcko D J Livingston 《The Journal of biological chemistry》1992,267(23):16029-16032
The mechanism of FK506 immunosuppression has been proposed to proceed by formation of a tight-binding complex with the intracellular 12-kDa FK506-binding protein (FKBP12). The FK506-FKBP12 complex then acts as a specific high-affinity inhibitor of the intracellular protein phosphatase PP2B (calcineurin), interrupting downstream dephosphorylation events required for T-cell activation. Site-directed mutagenesis of many of the surface residues of FKBP12 has no significant effect on its affinity for calcineurin. We have identified, however, three FKBP12 surface residues (Asp-37, Arg-42, and His-87) proximal to a solvent-exposed segment of bound FK506 that may be direct contacts in the calcineurin complex. Site-directed mutagenesis of two of these residues decreases the affinity of FKBP12-FK506 for calcineurin (Ki) from 6 nM for wild-type FKBP12 to 3.7 microM for a R42K/H87V double mutant, without affecting the peptidylprolyl isomerase activity or FK506 affinity of the mutant protein. These FKBP12 mutations along with several substitutions on FK506 known to affect calcineurin binding form a roughly 100-A2 region of the FKBP12-FK506 complex surface that is likely to be within the calcineurin binding site. 相似文献
19.
Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein. 总被引:18,自引:5,他引:18
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Y Koltin L Faucette D J Bergsma M A Levy R Cafferkey P L Koser R K Johnson G P Livi 《Molecular and cellular biology》1991,11(3):1718-1723