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BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Probing protein-protein interactions in living cells is crucial for understanding the protein functions and developing drugs. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time through live-cell imaging. A repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. The potential and utility of our approach was demonstrated by tracking the rapamycin-mediated interaction between FKBP12-rapamycin binding (FRB) domain and a FK506-binding protein (FKBP) and their localization by live-cell imaging. The present approach can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2015 Wiley Periodicals, Inc.)

Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research.

Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A2 (iPLA2) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis.

The tight complexes FKBP12 forms with immunosuppressive drugs, such as FK506 and rapamycin, are frequently used as models for developing approaches to structure-based drug design. Although the interfaces between FKBP12 and these ligands are well-defined structurally and are almost identical in the X-ray crystallographic structures of various complexes, our nuclear magnetic resonance studies have revealed the existence of substantial large-amplitude motions in the FKBP12-ligand interfaces that depend on the nature of the ligand. We have monitored these motions by measuring the rates of Tyr and Phe aromatic ring flips, and hydroxyl proton exchange for residues clustered within the FKBP12-ligand interface. The results show that the rates of hydroxyl proton exchange and ring flipping for Tyr26 are much slower in the FK506 complex than in the rapamycin complex, whereas the rates of ring flipping for Phe48 and Phe99 are significantly faster in the FK506 complex than in the rapamycin complex. The apparent rate differences observed for the interfacial aromatic residues in the two complexes confirm that these dynamic processes occur without ligand dissociation. We tentatively attribute the differential interface dynamics for these complexes to a single hydrogen bond between the ζ-hydrogen of Phe46 and the C32 carbonyl oxygen of rapamycin, which is not present in the KF506 complex. This newly identified Phe46 ζ-hydrogen bond in the rapamycin complex imposes motional restriction on the surrounding hydrophobic cluster and subsequently regulates the dynamics within the protein-ligand interface. Such information concerning large-amplitude dynamics at drug-target interfaces has the potential to provide novel clues for drug design.

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled., (Copyright © 2015 John Wiley & Sons, Inc.)

The function of the mammalian target of rapamycin (mTOR) is upregulated in response to cell stimulation with growing and differentiating factors. Active mTOR controls cell proliferation, differentiation and death. Since mTOR associates with different proteins to form two functional macromolecular complexes, we aimed to investigate the role of the mTOR1 and mTOR2 complexes in MEG-01 cell physiology in response to thrombopoietin (TPO). By using mTOR antagonists and overexpressing FKBP38, we have explored the role of both mTOR complexes in proliferation, apoptosis, maturation-like mechanisms, endoplasmic reticulum-stress and the intracellular location of both active mTOR complexes during MEG-01 cell stimulation with TPO. The results demonstrate that mTOR1 and mTOR2 complexes play different roles in the physiology of MEG-01 cells and in the maturation-like mechanisms; hence, these findings might help to understand the mechanism underlying generation of platelets.

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.

Background: Ligands for FK506-binding proteins, also referred to as neuroimmunophilin ligands, have repeatedly been described as neuritotrophic, neuroprotective or neuroregenerative agents. However, the precise molecular mechanism of action underlying the observed effects has remained elusive, which eventually led to a reduced interest in FKBP ligand development., Scope of Review: A survey is presented on the pharmacology of neuroimmunophilin ligands, of the current understanding of individual FKBP homologs in neuronal processes and an assessment of their potential as drug targets for CNS disorders., Major Conclusions: FKBP51 is the major target accounting for the neuritotrophic effect of neuroimmunophilin ligands. Selectivity against the homolog FKBP52 is essential for optimal neuritotrophic efficacy., General Significance: Selectivity within the FKBP family, in particular selective inhibition of FKBP12 or FKBP51, is possible. FKBP51 is a pharmacologically tractable target for stress-related disorders. The role of FKBPs in neurodegeneration remains to be clarified. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1(R206H)) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1(R206H) mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1(R206H) causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP., (Copyright © 2015, American Association for the Advancement of Science.)

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis-isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans-conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates.

Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca(2+)-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose-response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: Mechanistic/mammalian target of rapamycin (mTOR) activation by μ-opioid receptor (OPRM1) participates in antinociceptive tolerance, hyperalgesia, and physical dependence. Our previous study also showed that mTOR activation by OPRM1 could attenuate β amyloid oligomers-induced neurotoxicity. OPRM1 is demonstrated to interact with FK506-binding protein 12 (FKBP12). It is our great interest to investigate whether OPRM1-mediated mTOR signaling is related to receptor-FKBP12 association., Methods: The activities of mTOR and its downstream effector p70 S6K were measured by immunoblotting their phosphorylation status. The interaction of receptor with mTOR was detected by co-immunoprecipitation and immunofluorescence., Results: OPRM1 activation by morphine-induced time-dependent mTOR activation. PI3K-specific inhibitor LY294002 only blocked the late phase of mTOR activation. However, morphine-induced mTOR activation was totally blocked at all time points in cells expressing FKBP12 association-deficient mutant receptor. FKBP12 knockdown also blocked morphine-induced mTOR activation. Further analysis demonstrated that morphine treatment enhanced the association of receptor with phosphorylated mTOR, whereas decreased association was observed after FKBP12 knockdown, mTOR inhibition or in cells expressing FKBP12 association-deficient mutant., Conclusions: OPRM1-FKBP12 association played a key role in OPRM1-mediated mTOR activation, which could underlie the mechanisms of multiple physiological and pathological processes. Thus, our findings provide new avenue to modulating these processes., (© 2015 John Wiley & Sons Ltd.)

The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression.

The development of effective pharmacological inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-molecule antagonists disrupt the activity of only one domain in the target protein. We devised a chemical strategy that promotes ligand-dependent target protein degradation using as an example the transcriptional coactivator BRD4, a protein critical for cancer cell growth and survival. We appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compound, dBET1, induced highly selective cereblon-dependent BET protein degradation in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degradation of the cytosolic protein FKBP12. This chemical strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins., (Copyright © 2015, American Association for the Advancement of Science.)

The 12- and 12.6-kDa FK506-binding proteins, FKBP12 (12-kDa FK506-binding protein) and FKBP12.6 (12.6-kDa FK506-binding protein), have been implicated in the binding to and the regulation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), both tetrameric intracellular Ca2+-release channels. Whereas the amino acid sequences responsible for FKBP12 binding to RyRs are conserved in IP3Rs, FKBP12 binding to IP3Rs has been questioned and could not be observed in various experimental models. Nevertheless, conservation of these residues in the different IP3R isoforms and during evolution suggested that they could harbour an important regulatory site critical for IP3R-channel function. Recently, it has become clear that in IP3Rs, this site was targeted by B-cell lymphoma 2 (Bcl-2) via its Bcl-2 homology (BH)4 domain, thereby dampening IP3R-mediated Ca2+ flux and preventing pro-apoptotic Ca2+ signalling. Furthermore, vice versa, the presence of the corresponding site in RyRs implied that Bcl-2 proteins could associate with and regulate RyR channels. Recently, the existence of endogenous RyR-Bcl-2 complexes has been identified in primary hippocampal neurons. Like for IP3Rs, binding of Bcl-2 to RyRs also involved its BH4 domain and suppressed RyR-mediated Ca2+ release. We therefore propose that the originally identified FKBP12-binding site in IP3Rs is a region critical for controlling IP3R-mediated Ca2+ flux by recruiting Bcl-2 rather than FKBP12. Although we hypothesize that anti-apoptotic Bcl-2 proteins, but not FKBP12, are the main physiological inhibitors of IP3Rs, we cannot exclude that Bcl-2 could help engaging FKBP12 (or other FKBP isoforms) to the IP3R, potentially via calcineurin.

Once opened, ryanodine receptors (RyR) are efficient pathways for the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR). The precise nature of the Ca2+-release event, however, requires fine-tuning for the specific process and type of cell involved. For example, the spatial organization of RyRs, the luminal [Ca2+] and the influence of soluble regulators that fluctuate under physiological and pathophysiological control mechanisms, all affect the amplitude and duration of RyR Ca2+ fluxes. Various proteins are docked tightly to the huge bulky structure of RyR and there is growing evidence that, together, they provide a sophisticated and integrated system for regulating RyR channel gating. This review focuses on those proteins that are relevant to phosphorylation of RyR channels with particular reference to the cardiac isoform of RyR (RyR2). How phosphorylation of RyR affects channel activity and whether proteins such as the FK-506 binding proteins (FKBP12 and FKBP12.6) are involved, have been highly controversial subjects for more than a decade. But that is expected given the large number of participating proteins, the relevance of phosphorylation in heart failure and inherited arrhythmic diseases, and the frustrations of predicting relationships between structure and function before the advent of a high resolution structure of RyR.

An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

Motivation: Using molecular similarity to discover bioactive small molecules with novel chemical scaffolds can be computationally demanding. We describe Ultra-fast Shape Recognition with Atom Types (UFSRAT), an efficient algorithm that considers both the 3D distribution (shape) and electrostatics of atoms to score and retrieve molecules capable of making similar interactions to those of the supplied query., Results: Computational optimization and pre-calculation of molecular descriptors enables a query molecule to be run against a database containing 3.8 million molecules and results returned in under 10 seconds on modest hardware. UFSRAT has been used in pipelines to identify bioactive molecules for two clinically relevant drug targets; FK506-Binding Protein 12 and 11β-hydroxysteroid dehydrogenase type 1. In the case of FK506-Binding Protein 12, UFSRAT was used as the first step in a structure-based virtual screening pipeline, yielding many actives, of which the most active shows a KD, app of 281 µM and contains a substructure present in the query compound. Success was also achieved running solely the UFSRAT technique to identify new actives for 11β-hydroxysteroid dehydrogenase type 1, for which the most active displays an IC50 of 67 nM in a cell based assay and contains a substructure radically different to the query. This demonstrates the valuable ability of the UFSRAT algorithm to perform scaffold hops., Availability and Implementation: A web-based implementation of the algorithm is freely available at http://opus.bch.ed.ac.uk/ufsrat/.

Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.

The ryanodine receptors (RyRs) are high-conductance intracellular Ca(2+) channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant [Formula: see text] of this intermediate should be decreased, resulting in an increase of the affinity constant [Formula: see text]. The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor-1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was -10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR., (© 2014 Society for Laboratory Automation and Screening.)

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.

Unlabelled: In human cells TORC1 mTOR (target of rapamycin) protein kinase complex renders heat shock transcription factor 1 (Hsf1) competent for stress activation. In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 "bypass" strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9--the latter the ortholog of human S6 protein kinase 1., Significance: A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a "pro-survival" role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved.

The identification of target proteins of bioactive small molecules as bioprobe candidates or drug seeds is indispensable for elucidating their actions and predicting their side effects. To meet the current need, we developed a scheme for detection and identification of target proteins by using ribosome display and photo-cross-linking techniques, and demonstrated the feasibility. The mRNAs encoding full-length human proteins (FHPs) were constructed and translated in vitro to prepare pools of FHP-ribosome-mRNA complexes used for ribosome display selection. Expression levels of the FHPs were confirmed by Western blot analysis, and photo-cross-linked small-molecule beads were assessed through cell-free synthesized FHP binding assay. After ribosome display selection against photo-cross-linked small-molecule beads, RT-PCR using mRNAs recovered from the selected ternary complexes and electrophoresis of the PCR products allowed specific detection of the target proteins binding to the beads. In addition, a repeat of ribosome display selection enabled us to identify the target proteins even if the molar quantity was one ten-thousandth of that of the other proteins in a cell-free synthesized FHP pool. Therefore, these results showed that ribosome display using photo-cross-linked small-molecule beads and further extended FHP pool could be one of the powerful techniques for identification of unknown target proteins of bioactive small molecules.

The target of rapamycin (TOR) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown. We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum. Biological, genetic and biochemical functions of each component were investigated. The FgFkbp12-rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F. graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F. graminearum, a complex not observed in the yeast model. Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F. graminearum., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Molecular dynamics (MD) simulations in explicit solvent allow studying receptor-ligand binding processes including full flexibility of the binding partners and an explicit inclusion of solvation effects. However, in MD simulations, the search for an optimal ligand-receptor complex geometry is frequently trapped in locally stable non-native binding geometries. A Hamiltonian replica-exchange (H-REMD)-based protocol has been designed to enhance the sampling of putative ligand-receptor complexes. It is based on softening nonbonded ligand-receptor interactions along the replicas and one reference replica under the control of the original force field. The efficiency of the method has been evaluated on two receptor-ligand systems and one protein-peptide complex. Starting from misplaced initial docking geometries, the H-REMD method reached in each case the known binding geometry significantly faster than a standard MD simulation. The approach could also be useful to identify and evaluate alternative binding geometries in a given binding region with small relative differences in binding free energy.

Characterization of small molecule (SM)-protein interaction is of high importance in biomedical research such as target identification and proteomic profiling. Photo-cross-linking is a powerful and straightforward strategy to covalently capture SM's binding proteins. The DNA-based photoaffinity labeling method is able to capture SM's protein targets with high specificity but suffers low cross-linking efficiency, which limits its utility for low abundance and low affinity proteins. After screening a variety of cross-linkers, by utilizing the multivalency effect, the cross-linking efficiency was improved by nearly 7-fold without compromising probe specificity. The generality and performance of multivalent photoaffinity probes have been validated with a variety of SM-protein pairs in the complexity of cell lysates.

Site specific recombinases are frequently used as gene switches in transgenic animals where recombination is induced by drug treatment or by tissue specific recombinase expression. Alternatively, lentiviral gene transfer can be utilized for the genetic modification of a wide variety of cell types, albeit systems for tight control of transcriptional activity are scarce. Here, we combined lentiviral gene transfer and the development of a tightly drug-controlled FLP recombinase for the construction of "All-in-One" inducible gene expression systems. Tight control of FLP activity was achieved through N-terminal fusion with a FKBP12-derived conditional destruction domain and a C-terminal estrogen receptor binding domain making recombination dependent on the presence of Shield-1 and 4-hydroxytamoxifen. Exploiting the capacity of FLP to mediate excision and inversion, "All-in-One" lentiviral gene switch vector systems were generated where drug-induced recombination resulted in abrogation of FLP expression and subsequent overexpression or knockdown of the prototypical tumor suppressor phosphatase and tensin homolog PTEN. "All-in-One" vectors proved their functionality in a variety of hematopoietic cell lines, and primary murine bone marrow cells. Our new vector system thus combines the ease of lentiviral gene transfer and the power of site specific recombinases for analysis of gene function., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background & Aims: Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored., Methods: We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays., Results: VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9., Conclusions: In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Agonist-activated μ-opioid receptor (OPRM1) undergoes robust receptor phosphorylation by G protein-coupled receptor kinases and subsequent β-arrestin recruitment, triggering receptor internalization and desensitization. Morphine, a widely prescribed opioid, induces receptor phosphorylation inefficiently. Previously we reported that FK506 binding protein 12 (FKBP12) specifically interacts with OPRM1 and such interaction attenuates receptor phosphorylation and facilitates morphine-induced recruitment and activation of protein kinase C. In the current study, we demonstrated that the association of FKBP12 with OPRM1 also affects morphine-induced receptor internalization and G protein-dependent adenylyl cyclase desensitization. Morphine induced faster receptor internalization and adenylyl cyclase desensitization in cells expressing OPRM1 with Pro(353) mutated to Ala (OPRM1P353A), which does not interact with FKBP12, or in the presence of FK506 which dissociates the receptor-FKBP12 interaction. Furthermore, knockdown of cellular FKBP12 level by siRNA accelerated morphine-induced receptor internalization and adenylyl cyclase desensitization. Our study further demonstrated that peptidyl prolyl cis-trans isomerase activity of FKBP12 probably plays a role in inhibition of receptor phosphorylation. In the view that internalized receptor recycles and thus counteracts the development of analgesic tolerance, receptor's association with FKBP12 could also contribute to the development of morphine tolerance through modulation of receptor trafficking., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

FKBP12 is an important target in the treatment of transplant rejection and is also a promising target for cancer and neurodegenerative diseases. We determined for two ligands of nanomolar affinity the set of parameters in the CHARMM force field. The fitting procedure was based on reproducing the quantum chemistry data (distances, angles, and energies). Since the dynamical behavior of such ligands strongly depends on the dihedral angles, care was taken to derive the corresponding parameters. Moreover, since each of the central core region of these two ligands is similar to other known ligands or drugs of other proteins, part at least of these parameters could also be useful for these other ligands., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The sarcoplasmic reticulum (SR) Ca(2+) release channel or ryanodine receptor (RyR) of the vertebrate heart is regulated by the FK506-binding proteins, FKBP12 and FKBP12.6. This study examines whether temperature-related changes in the SR function of fish hearts are associated with changes in FKBP12 expression. For this purpose, a polyclonal antibody against trout FKBP12 was used to compare FKPB12 expression in cold-acclimated (4 °C, CA) and warm-acclimated (18 °C, WA) rainbow trout (Oncorhynchus mykiss), burbot (Lota lota) and crucian carp (Carassius carassius) hearts. FKBP12 expression was modulated in a species- and tissue-specific manner. Temperature acclimation affected FKBP12 expression only in atrial tissue. Changes in the ventricular FKBP12 expression were not detected in any of the fish species. In the atria of rainbow trout and crucian carp, temperature acclimation produced opposite thermal responses: FKBP12 increased in the trout atrium and decreased in the crucian carp atrium under cold acclimation. In the burbot heart, chronic temperature changes did not affect cardiac FKBP12 levels. Expression of FKBP12 mRNA in rainbow trout and crucian carp hearts suggests that the transcript levels are higher in the ventricle than in the atrium and are elevated by cold acclimation in trout, but not in crucian carp. Since FKBP12 is known to increase the Ca(2+) sensitivity of cardiac RyRs and thereby the opening frequency of the Ca(2+) release channels, temperature-related changes in FKBP12 expression may modify the SR function in excitation-contraction coupling. The cold-induced increase in FKBP12 in the trout atrium and decrease in the crucian carp atrium are consistent with the previously noted increase and decrease, respectively, of SR Ca(2+) stores in cardiac contraction in these species.

Aims: Cardamonin has previously demonstrated that it had an antiproliferative effect on vascular smooth muscle cells by inhibiting the activity of mammalian target of rapamycin (mTOR). The antiproliferative effect and the possible mechanism of combining with mTOR of cardamonin were investigated on A549 cells., Main Methods: Cell proliferation, cell cycle and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. mTOR and 12 kDa FK506 binding protein (FKBP12) were transfected into A549 cells by Lipofectamine. Western blots were used to examine the mTOR expressions and its activities, and the expressions of 70 kDa ribosomal S6 kinase (p70S6K), FKBP12 and Interleukin-2 (IL-2), respectively., Key Findings: Treated with cardamonin, the proliferation of A549 cells was inhibited. Meanwhile, cell cycle was blocked and DNA synthesis was decreased whereas cell apoptosis was promoted, and the activation of mTOR and p70S6K was decreased by cardamonin. Transfected with mTOR or FKBP12, proliferation of A549 cells was increased. Rapamycin had a similar degree of effect on antiproliferation of both transfected cells. However, the antiproliferative effect of cardamonin on mTOR transfected cells was stronger than that on FKBP12 transfected cells. Both rapamycin and cardamonin decreased the phosphorylation of mTOR and p70S6K in two kinds of transfected cells. Cardamonin had no effect on the expression of FKBP12 and IL-2, whereas the expressions were decreased by rapamycin., Significance: Cardamonin inhibited proliferation and induced apoptosis of A549 cells via mTOR. It might directly interact with mTOR independently of binding with FKBP12., (Copyright © 2014 Elsevier Inc. All rights reserved.)

One of the pathological hallmarks of Alzheimer's disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A beta), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer's disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12E31Q/D32N/W59F) where the residues Glu(31), Asp(32), and Trp(59) were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12E31Q/D32N/W59F mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12E31Q/D32N/W59F activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu(31), Asp(32), and Trp(59) in FKBP12 and Gln(31), Asn(32), and Phe(59) in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid receptor (OPRM1) desensitization and tolerance development. It was recently shown that receptor phosphorylation by G protein-coupled receptor kinase regulates agonist-dependent selective signaling and that inefficient phosphorylation of OPRM1 leads to PKCε activation and subsequent responses. Here, we demonstrate that such receptor phosphorylation and PKCε activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro(353) residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced receptor phosphorylation at Ser(375). The morphine-induced PKCε activation and the recruitment of PKCε to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKCε. Further analysis of the receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the receptor. The blockade of the FKBP12 association with the receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro(353) to Ala resulted in a reduction in PKCε and calcineurin recruitment to the receptor signaling complex. The receptor-associated calcineurin modulates OPRM1 phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the receptor phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the phosphorylation of the receptor and triggers the recruitment and activation of PKCε.

Stabilized Wittig olefination holds great potential as a bioconjugation reaction. We demonstrate that the reaction of stabilized phosphorus ylides (or phosphonium salts) with aryl aldehydes is sufficiently robust to be used for live cell affinity isolation and fluorescence tagging of a protein, FKBP12.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca(2+) levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca(2+) levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.