Immunosuppressant FK506 Research Articles

Effects of immunosuppression after limb fracture in mice on nociceptive, cognitive, and anxiety-related outcomes.

Chronic pain is a common and problematic consequence of injuries with few proven methods for prevention or treatment. In addition to pain, functional limitations and neuropsychiatric changes such as cognitive impairment and anxiety worsen outcomes. To determine whether inhibiting activation of the adaptive immune response after limb fracture would reduce pain, functional loss, memory changes, and anxiety. These experiments used a murine tibial fracture/cast immobilization model that develops these adverse outcomes. Adaptive immunity was blocked using the immunosuppressant FK506 beginning at the time of fracture. The administration of FK506 reduced mechanical allodynia and hind limb unweighting for weeks after cast removal as well as nonevoked pain measures. Fracture was associated with working memory loss in the Y-maze assay in vehicle- but not FK506-treated mice. Object recognition memory was not improved with FK506 after fracture. Also, vehicle- but not FK506-treated mice developed an anxiety phenotype. Impaired running wheel performance after cast removal over the following 2 weeks was not improved with FK506 administration. In addition, FK506 treatment blocked Immunoglobulin M (IgM) accumulation in the skin of the fractured limbs, and hippocampal enhancement of matrix metalloproteinase-8 expression, a metalloproteinase associated with neuroplastic changes after injuries, was completely blocked. Taken together, our results show that blocking the adaptive immune response after limb trauma reduces the severity of nociceptive and biological changes. The same treatment may reduce the adverse consequences of anxiety and memory deficits using some measures, but other measures of memory are not affected, and activity is not enhanced.

Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during β-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during β-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

Impact of FKBP52 on cell proliferation and hormone-dependent cancers.

FK506 binding protein 52 (FKBP52) (gene name FKBP4) is a 52 kDa protein that belongs to the FKBP family; it binds to the immunosuppressant FK506 and has proline isomerase activity. In addition to its FK domain-containing peptidylprolyl isomerase activity, FKBP52 also acts as a cochaperone through the tetratricopeptide repeat domain that mediates binding to heat shock protein 90. Previous studies have reported that FKBP52 is associated with hormone-dependent, stress-related, and neurodegenerative diseases, revealing its diverse functions. In particular, the effects of FKBP52 on cancer have attracted considerable attention. FKBP52 promotes the growth of hormone-dependent cancers by activating steroid hormone receptors. Recent studies have shown that the expression of FKBP52 is increased not only in steroid hormone-dependent cancer cells but also in colorectal, lung, and liver cancers, revealing its diverse functions that contribute to cancer growth. This review summarizes reports related to hormone-dependent cancer and cell proliferation in terms of the structure of FKBP52 and its function on interacting molecules.

Natural product ligands of FKBP12: Immunosuppressive antifungal agents FK506, rapamycin, and beyond.

Natural product ligands of FKBP12: Immunosuppressive antifungal agents FK506, rapamycin, and beyond.

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

The Prognostic Significance of FKBP1A and Its Related Immune Infiltration in Liver Hepatocellular Carcinoma.

Liver hepatocellular carcinoma (LIHC) remains a global health challenge with poor prognosis and high mortality. FKBP1A was first discovered as a receptor for the immunosuppressant drug FK506 in immune cells and is critical for various tumors and cancers. However, the relationships between FKBP1A expression, cellular distribution, tumor immunity, and prognosis in LIHC remain unclear. Here, we investigated the expression level of FKBP1A and its prognostic value in LIHC via multiple datasets including ONCOMINE, TIMER, GEPIA, UALCAN, HCCDB, Kaplan-Meier plotter, LinkedOmics, and STRING. Human liver tissue microarray was employed to analyze the characteristics of FKBP1A protein including the expression level and pathological alteration in cellular distribution. FKBP1A expression was significantly higher in LIHC and correlated with tumor stage, grade and metastasis. The expression level of the FKBP1A protein was also increased in LIHC patients along with its accumulation in endoplasmic reticulum (ER). High FKBP1A expression was correlated with a poor survival rate in LIHC patients. The analysis of gene co-expression and the regulatory pathway network suggested that FKBP1A is mainly involved in protein synthesis, metabolism and the immune-related pathway. FKBP1A expression had a significantly positive association with the infiltration of hematopoietic immune cells including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Moreover, M2 macrophage infiltration was especially associated with a poor survival prognosis in LIHC. Furthermore, FKBP1A expression was significantly positively correlated with the expression of markers of M2 macrophages and immune checkpoint proteins such as PD-L1, CTLA-4, LAG3 and HAVCR2. Our study demonstrated that FKBP1A could be a potential prognostic target involved in tumor immune cell infiltration in LIHC.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

RationaleHuman coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively.MethodsPrimary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication.ResultsBoth CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model.ConclusionThe immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Development of a novel pharmacophore model to screen specific inhibitors for the serine-threonine protein phosphatase calcineurin

Calcineurin (CaN) is a calcium/calmodulin-dependent serine/threonine phosphatase with a crucial role in cellular homeostasis. It is also the target of the Food and Drug Administration (FDA) approved immunosuppressant drugs FK506 and cyclosporine A. Recent work from our group and others indicated that an uncontrolled increase in CaN activity causes synaptic dysfunction and neuronal death in various models of neurodegenerative diseases associated with calcium dysregulation. Furthermore, pharmacological normalization of CaN activity can prevent disease progression in animal models. However, none of the FDA-approved CaN inhibitors bind CaN directly, leading to adverse side effects. The development of direct CaN inhibitors is required to reduce off-target effects, but its highly conserved active site and similar mechanism of action with other protein serine/threonine phosphatases impose a significant challenge. In this work, we developed a novel pharmacophore model to screen for CaN-specific inhibitors. Then, we performed a virtual screen for molecules having the pharmacophore model. We also show that the molecules identified in this screen can inhibit CaN with a low micromolar IC50. Interestingly, the inhibitors identified from the screen do not inhibit phosphoprotein phosphatase 2A, a member of the serine/threonine phosphatase family that shares 43% sequence identity with the CaN active site. The pharmacophore model that we developed and validated in this work may help to accelerate the development of specific CaN inhibitors.

Systematic investigation and comparison of US FDA-approved immunosuppressive drugs FK506, cyclosporine and rapamycin for neuromuscular regeneration following chronic nerve compression injury.

Aim: To compare therapeutic benefits of different immunophilin ligands for treating nerve injuries. Materials & methods: Cyclosporine, FK506 and rapamycin, were evaluated first in vitro on a serum-free culture of embryonic dorsal root gangliafollowed by a new in vivo model of chronic nerve compression. Results: Outcomes of the in vitro study have shown a potent effect of cyclosporine and FK506, on dorsal root ganglia axonal outgrowth, comparable to the effect of nerve growth factor. Rapamycin exhibited only amoderate effect. The in vivo study revealed the beneficial effects of cyclosporine, FK506 and rapamycin for neuromuscular regeneration. Cyclosporine showed the better maintenance of the tissues and function. Conclusion: Cyclosporine, FK506 and rapamycin drugs showed potential for treating peripheral nerve chronic compression injuries.

Multi-Omics Analysis of the Expression and Prognosis for FKBP Gene Family in Renal Cancer.

BackgroundThe FK506-binding protein (FKBP) is a family of intracellular receptors that can bind specifically to the immunosuppressant FK506 and rapamycin. Although FKBPs play crucial roles in biological processes and carcinogenesis, their prognostic value and molecular mechanism in clear cell renal cell carcinoma (ccRCC) remain unclear.MethodsUsing pan-cancer data from The Cancer Genome Atlas (TCGA) and public databases, we analyzed the expression and correlation of FKBPs in 33 tumor types. Survival and Cox regression analyses were employed to explore the prognostic value of FKBPs. The relationship with tumor microenvironment and stemness indices was taken into account to evaluate the function of FKBPs. We constructed a risk score model to predict the prognosis of patients with ccRCC. The receiver operating characteristic (ROC) curve was performed to further test the prognostic ability of our model. Nomogram, joint effects analysis, and clinical relevance were performed to assist the clinician. Gene set enrichment analysis (GSEA) and cell line experiments were performed to investigate the function and molecular mechanisms of FKBPs in patients with ccRCC. Paired clinical specimens and multi-omics analysis were used to further validate and explore the factors affecting gene expression in ccRCC patients.ResultsThe expression levels of FKBP10 and FKBP11 were higher in ccRCC tissues than in normal tissues. The alteration in expression may be because of the degree of DNA methylation. Increased expression levels of FKBP10 and FKBP11 were associated with worse overall survival (OS). More importantly, GSEA revealed that FKBP10 is mainly involved in cell metabolism and autophagy, whereas FKBP11 is mainly associated with immune-related biological processes and autophagy. Cell Counting Kit 8 (CCK-8) and Transwell assays revealed that knockdown of FKBP10 and FKBP11 inhibits proliferation, migration, and invasion of the ccRCC cell line.ConclusionFKBP10 and FKBP11 play important roles in ccRCC phenotypes and are potential prognostic markers as well as new therapeutic targets for patients with ccRCC.

Local FK506 drug delivery enhances nerve regeneration through fresh, unprocessed peripheral nerve allografts

ObjectiveNerve allografts offer many advantages in the reconstruction of peripheral nerve gaps: they retain their native microstructure, contain pro-regenerative Schwann cells, are widely available, and avoid donor site morbidity. Unfortunately, clinical use of nerve allografts is limited by the need for systemic immunosuppression and its adverse effects. To eliminate the toxicity of the systemic immunosuppressant FK506, we developed a local FK506 drug delivery system (DDS) to provide drug release over 28 days. The study objective was to investigate if the local FK506 DDS enhances nerve regeneration in a rodent model of nerve gap defect reconstruction with immunologically-disparate nerve allografts. MethodsIn male Lewis rats, a common peroneal nerve gap defect was reconstructed with either a 20 mm nerve isograft from a donor Lewis rat or a 20 mm fresh, unprocessed nerve allograft from an immunologically incompatible donor ACI rat. After 4 weeks of survival, nerve regeneration was evaluated using retrograde neuronal labelling, quantitative histomorphometry, and serum cytokine profile. ResultsTreatment with both systemic FK506 and the local FK506 DDS significantly improved motor and sensory neuronal regeneration, as well as histomorphometric indices including myelinated axon number. Rats with nerve allografts treated with either systemic or local FK506 had significantly reduced serum concentrations of the pro-inflammatory cytokine IL-12 compared to untreated vehicle control rats with nerve allografts. Serum FK506 levels were undetectable in rats with local FK506 DDS. InterpretationThe local FK506 DDS improved motor and sensory nerve regeneration through fresh nerve allografts to a level equal to that of either systemic FK506 or nerve isografting. This treatment may be clinically translatable in peripheral nerve reconstruction or vascularized composite allotransplantation.

A Rice Immunophilin Homolog, OsFKBP12, Is a Negative Regulator of Both Biotic and Abiotic Stress Responses.

A class of proteins that were discovered to bind the immunosuppressant drug FK506, called FK506-binding proteins (FKBPs), are members of a sub-family of immunophilins. Although they were first identified in human, FKBPs exist in all three domains of life. In this report, a rice FKBP12 homolog was first identified as a biotic stress-related gene through suppression subtractive hybridization screening. By ectopically expressing OsFKBP12 in the heterologous model plant system, Arabidopsis thaliana, for functional characterization, OsFKBP12 was found to increase susceptibility of the plant to the pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). This negative regulatory role of FKBP12 in biotic stress responses was also demonstrated in the AtFKBP12-knockout mutant, which exhibited higher resistance towards Pst DC3000. Furthermore, this higher-plant FKBP12 homolog was also shown to be a negative regulator of salt tolerance. Using yeast two-hybrid tests, an ancient unconventional G-protein, OsYchF1, was identified as an interacting partner of OsFKBP12. OsYchF1 was previously reported as a negative regulator of both biotic and abiotic stresses. Therefore, OsFKBP12 probably also plays negative regulatory roles at the convergence of biotic and abiotic stress response pathways in higher plants.

Proof that the high molecular weight immunophilin FKBP52 mediates the in vivo neuroregenerative effect of the macrolide FK506

The immunosuppressant drug FK506 (or tacrolimus) is a macrolide that binds selectively to immunophilins belonging to the FK506-binding protein (FKBP) subfamily, which are abundantly expressed proteins in neurons of the peripheral and central nervous systems. Interestingly, it has been reported that FK506 increases neurite outgrowth in cell cultures, implying a potential impact in putative treatments of neurodegenerative disorders and injuries of the nervous system. Nonetheless, the mechanism of action of this compound is poorly understood and remains to be elucidated, with the only certainty that its neurotrophic effect is independent of its primary immunosuppressant activity. In this study it is demonstrated that FK506 shows efficient neurotrophic action in vitro and profound effects on the recovery of locomotor activity, behavioural features, and erectile function of mice that underwent surgical spinal cord injury. The recovery of the locomotor activity was studied in knock-out mice for either immunophilin, FKBP51 or FKBP52. The experimental evidence demonstrates that the neurotrophic actions of FK506 are the consequence of its binding to FKBP52, whereas FK506 interaction with the close-related partner immunophilin FKBP51 antagonises the function of FKBP52. Importantly, our study also demonstrates that other immunophilins do not replace FKBP52. It is concluded that the final biological response is the resulting outcome of the drug binding to both immunophilins, FKBP51 and FKBP52, the latter being the one that commands the dominant neurotrophic action in vivo.

Mandibular allotransplant procedures in dogs with the immunosuppressive agent FK506

ObjectiveTreatment of mandibular lesions sometimes leads to the formation of large defects in the bone. Mandible reconstruction using grafted tissue, bone, and/or medical implants is required following the resection of such lesions. However, such grafts may not completely restore the continuity of the mandible. Facial transplantations using allografts are common, but mandibular allotransplantation has not been performed in humans, due to the unstable immune reaction of the complex tissue. In this study, mandibular transplantations performed under the effect of the immunosuppressive agent, FK506, were evaluated in dogs. MethodsMandibular allotransplantations were performed in 21 pairs of sex-blind beagle dogs. The left side of the mandible in the donor dog was transplanted to the left side of the mandible in the recipient dog. The FK506 doses were 0 mg for 3 dog pairs, 0.08 mg/kg/day for 3, 0.12 mg/kg/day for 5, 0.16 mg/kg/day for 5, and 0.32 mg/kg/day for 5 pairs. Twelve weeks after transplantation, the dogs were euthanized. Computed tomography (CT) and histological examinations of the transplanted sides were performed. ResultsSuccessful graft acceptance was dependent on immunosuppressive agent doses, and dogs administered more than 0.16 mg/kg/day FK506 showed 100 % survival. However, dogs administered 0.32 mg/kg/day FK506 showed significant weight loss 12 weeks after the surgery. CT findings showed thick mandibular cortical bone in the transplanted sites. ConclusionsThese results indicate that 0.16 mg/kg/day FK506 was the ideal dose for allotransplantations. Bone hyperplasia was observed in the transplanted sides, but it is not a disadvantage for allotransplantations.

Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genescooperatively with Hmo1 in Saccharomyces cerevisiae.

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

Uncovering The Unique Functions And Regulation Of The Palmitoylated Calcineurin Isoform, CNβ1

Calcineurin (CN), the serine/threonine protein phosphatase and the target of immunosuppressants FK506 and CysA, is a key regulator of Ca2+ signaling with critical roles in the immune, cardiovascular and nervous systems. CN is a heterodimer of regulatory and catalytic subunits whose functions and regulation by Ca2+ and calmodulin are well understood for canonical CN isozymes (CNalpha, CNβ2). In contrast, the CNβ1 isozyme, contains a catalytic subunit with a non‐canonical C‐terminus generated by alternative 3’ pre‐mRNA processing. The few studies of CNβ1, conserved in eukaryotes and broadly expressed in human tissues, demonstrate its unique physiological functions. For example, overexpression of the CNβ2 promotes cardiac hypertrophy through activation of NFAT, whereas CNβ1 is cardioprotective and does not dephosphorylate NFAT. However, CNβ1 specific substrates remain unknown.We determined that the unique C‐tail confers distinct regulatory properties, intracellular localization and function to CNβ1. In vitro, CNβ1 displays distinct enzymatic properties. Instead of the auto‐inhibitory domain that blocks the active site of canonical CN isoforms under non‐signaling conditions, CNβ1 is autoinhibited by a sequence motif at its C` tail that blocks substrate binding. We show that CNβ1 localizes to the plasma membrane (PM), Golgi, and intracellular vesicles, in contrast to the cytosolic CNβ2, due to the palmitoylation of two conserved cysteine residues unique to its C‐tail. Palmitoylation allows CNβ1 to access substrates that are distinct from canonical CN isoforms. CNβ1 preferentially interacts with membrane proteins, and all members of a highly conserved PI4‐kinase complex, PI4KIIIA, TTC7B, FAM126A and EFR3B, were identified as CNβ1‐specific interactors by AP‐MS. This complex recruits the cytosolic PI4KIIIA to the PM where it synthesizes phosphatidylinositol‐4‐phosphate (PI4P), a precursor of the critical signaling phospholipid, PI(4,5)P2, required for sustained Ca2+ signaling through GPCRs. We identify a CN binding motif in FAM126A, mutation of which results in FAM126A hyperphosphorylation, and prevents association of CNβ1 with the PI4KIIIA complex. Using BRET‐based detection of phosphoinositides in live cells, we show that CN promotes PI4P synthesis at the PM during signaling through the muscarinic receptor, and hypothesize that CNβ1 mediates this regulation by dephosphorylating FAM126A at the PM.Using pulse‐chase analysis in cells metabolically labelled with a palmitate analog, we demonstrate that palmitoylation of CNβ1 is dynamic and turns over rapidly. We are currently investigating whether dynamic palmitoylation regulates localization and activity CNβ1 activity in vivo during signaling. Together, these studies provide the first mechanistic understanding of CNβ1 and not only uncovers a novel Ca2+‐dependent mechanism for activation of PI4P synthesis at the PM during signaling but also suggests palmitoylation as a novel mechanism that confers spatio/temporal regulation of calcineurin signaling in cells.Support or Funding InformationNIGMS

FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus

The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80’s loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40’s loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40’s and 50’s loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80’s loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.

Characterization of three FK506-binding proteins in the entomopathogenic fungus Beauveria bassiana

FK506 binding proteins (FKBPs) participate in regulation of diverse biological processes. However, the role of these proteins in insect-pathogenic fungi is far from well understood. To investigate the functions of FKBPs in Beauveria bassiana, a widely used entomopathogenic fungus for control of insect pests, we identify three putative FKBP genes, Bbfkbp12, Bbfkbp15, and Bbfkbp50, in the fungus. Gene-disruption experiments show that loss of Bbfkbp12 results in a significant increase of resistance of B. bassiana against the immunosuppressive compounds FK506 and rapamycin, while loss of Bbfkbp50 leads to the resistance to the ergosterol synthesis inhibitor lovastatin. Transcription assays of calcineurin (CaN)- and mTOR (mammalian target of rapamycin)-downstream target genes confirm that BbFKBP12 is the target of both FK506 and rapamycin, associated with CaN- and mTOR-signal pathways in B. bassiana. GFP-tagging of the proteins shows that BbFKBP12 and BbFKBP15 localize in cytoplasm while BbFKBP50 in nucleus. Our results provide useful information for the study of functions of CaN- and mTOR-mediated signaling, and ergosterol synthesis in the entomopathogenic fungi.

FK506 regulates Ca2+ release evoked by inositol 1,4,5-trisphosphate independently of FK-binding protein in endothelial cells.

Background and PurposeFK506 and rapamycin are modulators of FK‐binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side‐effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 receptors (IP3R) and ryanodine receptors (RyR) to alter Ca2+ signalling in endothelial cells.Experimental ApproachWe investigated the effects of FK506 and rapamycin on Ca2+ release via IP3R and RyR in hundreds of endothelial cells, using the indicator Cal‐520, in intact mesenteric arteries from male Sprague‐Dawley rats. IP3Rs were activated by acetylcholine or localised photo‐uncaging of IP3, and RyR by caffeine.Key ResultsWhile FKBPs were present, FKBP modulation with rapamycin did not alter IP3‐evoked Ca2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP3‐evoked Ca2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP3‐evoked Ca2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP3‐evoked Ca2+ release. These findings suggest that IP3‐evoked Ca2+ release is not modulated by FKBP, but by FK506‐mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca2+ signalling suggesting that RyR is not functional in native endothelium.Conclusion and ImplicationsThe hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca2+ release and altered FKBP binding to IP3 and RyR.