Solution structure of FK506-binding protein 12 from Aedes aegypti

Dengue remains one of the major public concerns as the virus eludes the immune response. Currently, no vaccines or antiviral therapeutics are available for dengue prevention or treatment. Immunosuppressive drug FK506 shows an antimalarial activity, and its molecular target, FK506‐binding protein (FKBP), was identified in human Plasmodium parasites. Likewise, a conserved FKBP family protein has also been identified in Aedes aegypti (AaFKBP12), which is expected to play a similar role in the life cycle of Aedes aegypti, the primary vector of dengue virus infection. As FKBPs belong to a highly conserved class of immunophilin family and are involved in key biological regulations, they are considered as attractive pharmacological targets. In this study, we have determined the nuclear magnetic resonance solution structure of AaFKBP12, a novel FKBP member from Aedes aegypti, and presented its structural features, which may facilitate the design of potential inhibitory ligands against the dengue‐transmitting mosquitoes. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

FKBP133: a novel mouse FK506-binding protein homolog alters growth cone morphology

FK506-binding proteins are the peptidyl prolyl cis-trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott-Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.     

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.     

Evolution of the 12 kDa FK506-binding protein gene

Background information. The FKBPs (FK506‐binding proteins) belong to a ubiquitous family of proteins that are found in a wide range of taxonomic groups. These proteins participate in a variety of pathways, including protein folding, down‐regulation of T‐cell activation and inhibition of cell‐cycle progression. Results. A cDNA encoding the 12 kDa FKBP gene orthologue (FKBP12) in Bombyx mori was been isolated from both Bm‐5 cultured cells and silk‐gland tissue. Using the FKBP12 cDNA in combination with the B. mori 6× whole‐genome shotgun database, we were able to identify the FKBP12 gene, as well as the positions of its intron—exon junctions. Conclusions. FKBP12 exon sizes and intronic positions are highly conserved among FKBP12 orthologues in 24 diverse genomes. Comparison of 41 FKBP12 genes revealed several intronic insertion and deletion events throughout evolution. In addition, paralogous FKBP12 isoforms were identified in all 12 vertebrate genomes. Both structural and phylogenetics analyses suggest that the isoforms may be evolving independently, possibly due to the distinct functional roles played by each paralogue.     

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (K _d=0.4 nM) and rapamycin (K _d=0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFVLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other post-translational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaertet al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent -strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.     

Genome-wide identification and analysis of FK506-binding protein gene family in peach (Prunus persica)

The FKBP protein family has prolyl isomerase activity and is related in function to cyclophilins. FKBPs are known to be involved in many biological processes including hormone signaling, plant growth, and stress responses through a chaperone or an isomerization of proline residues during protein folding. The availability of complete peach genome sequences allowed the identification of 21 FKBP genes by HMMER and BLAST analyses. Scaffold locations of these FKBP genes in the peach genome were determined and the protein domain and motif organization of peach FKBPs were analyzed. The phylogenetic relationships between peach FKBPs were also assessed. The expression profiles of peach FKBP gene results revealed that most peach FKBPs were expressed in all tissues, while a few peach FKBPs were specifically expressed in some of the tissues. This data could contribute to better understanding of the complex regulation of the peach FKBP gene family, and also provide valuable information for further research in peach functional genomics.     

Genome-wide identification and analysis of FK506-binding protein family gene family in strawberry (Fragaria × ananassa)

The FK506 binding proteins (FKBPs) are abundant and ubiquitous proteins belonging to the large peptidyl-prolylcis-trans isomerase superfamily. FKBPs are known to be involved in many biological processes including hormone signaling, plant growth, and stress responses through a chaperone or an isomerization of proline residues during protein folding. The availability of complete strawberry genome sequences allowed the identification of 23 FKBP genes by HMMER and blast analysis. Chromosome scaffold locations of these FKBP genes in the strawberry genome were determined and the protein domain and motif organization of FaFKBPs analyzed. The phylogenetic relationships between strawberry FKBPs were also assessed. The expression profiles of FaFKBPs genes results revealed that most FaFKBPs were expressed in all tissues, while a few FaFKBPs were specifically expressed in some of the tissues. These data not only contribute to some better understanding of the complex regulation of the strawberry FKBP gene family, but also provide valuable information for further research in strawberry functional genomics.     

A nuclear FK506-binding protein is a histone chaperone regulating rDNA silencing

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.     

Genome-wide analysis and environmental response profiling of the FK506-binding protein gene family in maize (Zea mays L.)

The FK506-binding proteins (FKBPs) belong to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, and have been implicated in a wide spectrum of biological processes, including protein folding, hormone signaling, plant growth, and stress responses. Genome-wide structural and evolutionary analyses of the entire FKBP gene family have been conducted in Arabidopsis and rice. In the present study, a genome-wide analysis was performed to identify all maize FKBP genes. The availability of complete maize genome sequences allowed for the identification of 24 FKBP genes. Chromosomal locations in the maize genome were determined and the protein domain and motif organization of ZmFKBPs analyzed. The phylogenetic relationships between maize FKBPs were also assessed. The expression profiles of ZmFKBP genes were measured under different environmental conditions and revealed distinct ZmFKBP gene expression patterns under heat, cold, salt, and drought stress. These data not only contribute to a better understanding of the complex regulation of the maize FKBP gene family, but also provide evidence supporting the role of FKBPs in multiple signaling pathways involved in stress responses. This investigation may provide valuable information for further research on stress tolerance in plants and potential strategies for enhancing maize survival under stressful conditions.     

Comparative analysis of different peptidyl-prolyl isomerases reveals FK506-binding protein 12 as the most potent enhancer of alpha-synuclein aggregation

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.     

A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein

A member of a eukaryotic gene superfamily, encoding a peptidylproline cis-trans-isomerase (rotamase) has been isolated from a maize (Zea mays L. A69Y+) endosperm cDNA library. The maize sequence (mzFKBP-66) encodes a 66-kDa polypeptide most closely related to the subclass of rotamases which bind an immunosuppressive drug, FK506, (termed FK506-binding proteins, FKBPs), and possesses four tandem copies of the FKBP-like binding domain. The sequence mzFKBP-66 is expressed ubiquitously in the maize plant, and the protein encoded is present in both cytosolic and nuclear compartments within the cell. Both the native mzFKBP-66 and a recombinant protein overexpressed in Escherichia coli showed peptidylproline␣cis-trans-isomerase (PPIase) activity at rates comparable to those reported for mammalian immunophilins. This activity was also sensitive to inhibition by FK506. Immunoaffinity chromatography using anti-mzFKBP66 demonstrated an association of the protein with an unknown 36-kDa polypeptide, and affinity chromatography of mzFKBP-66 on calmodulin-agarose beads indicated the presence of a calmodulin-binding site. The existence of mzFKBP-66-associated proteins suggests that plant immunophilins may act as part of multicomponent complexes, as has been shown for other representatives of this class of enzyme. Received: 9 June 1997 / Accepted: 19 August 1997     

Escherichia coli and other species of the enterobacteriaceae encode a protein similar to the family of Mip-like FK506-binding proteins

A newly identified gene in Escherichia coli, fkpA, encodes a protein with extensive similarity to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. The FkpA protein may be a new member of the family of FK506-binding proteins (FKBPs) because its carboxyl domain includes a sequence that matches the consensus FK506-binding motif in 40 of 48 positions. including those amino acids at the active site that form hydrogen bonds with the drug FK506. The amino acid sequence of the 29kDa FkpA protein is 30–35% identical to the Mip proteins of L. pneumophila, L. micdadei, and C. trachomatis. Of the 270 amino acids of FkpA, 113 (42%) are identical to the sequence of one or another of these Mip proteins. Overexpression of FkpA or deletion of fkpA from the E. coli chromosome had no detrimental effect on bacterial growth, indicating that fkpA is not an essential gene. Hybridization of fkpA-specific DNA probes to genomic blots révealed that similar genes exist in several representatives of the Enterobacteriaceae. Thus, mip-like genes are not found exelusively in bacteria having a predominately intracellular life style, but instead appear to be a new FKBP subfamily that is a common constituent of many bacteria.     

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug‐resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X‐ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.     

A calcineurin antifungal strategy with analogs of FK506

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.     

FK506-binding protein (FKBP12) regulates ryanodine receptor-evoked Ca2+ release in colonic but not aortic smooth muscle

In smooth muscle, the ryanodine receptor (RyR) mediates Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store. Release may be regulated by the RyR accessory FK506-binding protein (FKBP12) either directly, as a result of FKBP12 binding to RyR, or indirectly via modulation of the activity of the phosphatase calcineurin or kinase mTOR. Here we report that RyR-mediated Ca(2+) release is modulated by FKBP12 in colonic but not aortic myocytes. Neither calcineurin nor mTOR are required for FKBP12 modulation of Ca(2+) release in colonic myocytes to occur. In colonic myocytes, co-immunoprecipitation techniques established that FKBP12 and calcineurin each associated with the RyR2 receptor isoform (the main isoform in this tissue). Single colonic myocytes were voltage clamped in the whole cell configuration and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) increases evoked by the RyR activator caffeine. Under these conditions FK506, which displaces FKBP12 (to inhibit calcineurin) and rapamycin, which displaces FKBP12 (to inhibit mTOR), each increased the [Ca(2+)](c) rise evoked by caffeine. Notwithstanding, neither mTOR nor calcineurin are required to potentiate caffeine-evoked Ca(2+) increases evoked by each drug. Thus, the mTOR and phosphatidylinositol 3-kinase inhibitor, LY294002, which directly inhibits mTOR without removing FKBP12 from RyR, did not alter caffeine-evoked [Ca(2+)](c) transients. Nor did inhibition of calcineurin by cypermethrin, okadaic acid or calcineurin inhibitory peptide block the FK506-induced increase in RyR-mediated Ca(2+) release. In aorta, although RyR3 (the main isoform), FKBP12 and calcineurin were each present, RyR-mediated Ca(2+) release was unaffected by either FK506, rapamycin or the calcineurin inhibitors cypermethrin and okadaic acid in single voltage clamped aortic myocytes. Presumably failure of FKBP12 to associate with RyR3 resulted in the immunosuppressant drugs (FK506 and rapamycin) being unable to alter the activity of RyR. The effects of these drugs are therefore, apparently dependent on an association of FKBP12 with RyR. Together, removal of FKBP12 from RyR augmented Ca(2+) release via the channel in colonic myocytes. Neither calcineurin nor mTOR are required for the FK506- or rapamycin-induced potentiation of RyR Ca(2+) release to occur. The results indicate that FKBP12 directly inhibits RyR channel activity in colonic myocytes but not in aorta.     

Mechanism of osteogenic induction by FK506 via BMP/Smad pathways

FK506 is an immunosuppressant that exerts effects by binding to FK506-binding protein 12 (FKBP12). Recently, FK506 has also been reported to promote osteogenic differentiation when administered locally or in vitro in combination with bone morphogenetic proteins (BMPs), although the underlying mechanism remains unclarified. The present study initially showed that FK506 alone at a higher concentration (1muM) induced osteogenic differentiation of mesenchymal cell lines, which was suppressed by adenoviral introduction of Smad6. FK506 rapidly activates the BMP-dependent Smads in the absence of BMPs, and the activation was blocked by Smad6. Overexpression of FKBP12, which was reported to block the ligand-independent activation of BMP type I receptor A (BMPRIA), suppressed Smad signaling induced by FK506, but not that induced by BMP2. BMPRIA and FKBP12 bound to each other, and this binding was suppressed by FK506. These data suggest that FK506 promotes osteogenic differentiation by activating BMP receptors through interacting with FKBP12.     

FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T

Aggregation of alpha-synuclein (α-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of α-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P α-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants α-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.