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FKBP4, a member of the FK506-binding protein (FKBP) family, is a promising target for a variety of disorders, including cancer. However, its underlying molecular mechanism and potential function in hepatocellular carcinoma (HCC) are largely elusive. Therefore, we aimed to investigate the expression status, functional implications and underlying mechanisms of FKBP4 in HCC. Our bioinformatics analysis of TCGA LIHC datasets, ICGC LIRI-JP datasets and GEO datasets results showed FKBP4 was upregulated in HCC tissues. We also confirmed the elevated FKBP4 in clinical HCC samples. Additionally, quantitative RT-PCR results revealed FKBP4 was highly expressed in all five tested HCC cell lines. We also observed a correlation between elevated FKBP4 expression and poor prognosis in HCC patients. Loss of FKBP4 can inhibit the proliferation and migration in HCC cells. Furthermore, we found that silencing FKBP4 suppressed glucose uptake, lactic acid production and  18 F-FDG uptake compared with the control group. Mechanistically, our funding indicated that FKBP4 participates in glycolysis through p53 mediated HK2 signaling pathway, specially, FKBP4 knockdown promotes the expression and stability of p53 protein rather than affecting the transcription level. Finally, rescue experiments revealed that simultaneous knockdown of both FKBP4 and p53 partially reversed the inhibitory effects on HK2 protein levels and  18 F-FDG uptake. Our study elucidates a novel role of FKBP4 in promoting HCC development and glycolysis by modulating the p53/HK2 signaling pathway. Given the critical role of aerobic glycolysis in the progression of HCC, targeting FKBP4 may offer a new therapeutic strategy for treating this malignancy., (© 2024. The Author(s).)

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection., (© 2024. The Author(s).)

Post-traumatic stress disorder (PTSD) leads to enhanced alcohol drinking and development of alcohol use disorder (AUD). Identifying shared neural mechanisms might help discover new therapies for PTSD/AUD. Here, we employed a rat model of comorbid PTSD/AUD to evaluate compounds that inhibit FK506-binding protein 51 (FKBP5), a co-chaperone modulator of glucocorticoid receptors implicated in stress-related disorders. Male and female rats received a familiar avoidance-based shock stress followed by voluntary alcohol drinking. We then assessed trauma-related behaviors through sleep bout cycles, hyperarousal, fear overgeneralization, and irritability. To evaluate the role of stress and alcohol history on the sensitivity to FKBP5 inhibitors, in two separate studies, we administered two FKBP5 inhibitors, benztropine (Study 1) or SAFit2 (Study 2). FKBP5 inhibitors were administered on the last alcohol drinking session and prior to each trauma-related behavioral assessment. We also measured plasma corticosterone to assess the actions of FKBP5 inhibitors after familiar shock stress and alcohol drinking. Benztropine reduced alcohol preference in stressed males and females, while aggressive bouts were reduced in benztropine-treated stressed females. During hyperarousal, benztropine reduced several startle response outcomes across stressed males and females. Corticosterone was reduced in benztropine-treated stressed males. The selective FKBP5 inhibitor, SAFit2, reduced alcohol drinking in stressed males but not females, with no differences in irritability. Importantly, SAFit2 decreased fear overgeneralization in stressed males and females. SAFit2 also reduced corticosterone across stressed males and females. Neither FKBP5 inhibitor changed sleep bout structure. These findings indicate that FKBP5 inhibitors modulate stress-related alcohol drinking and partially modulate trauma-related behaviors. This work supports the hypothesis that targeting FKBP5 may alleviate PTSD/AUD comorbidity., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

We investigated gluteal (GSAT) and abdominal subcutaneous adipose tissue (ASAT) DNA methylation of FKBP5 in response to a 12-week intervention in African women with obesity, as well as the effect of the rs1360780 single nucleotide polymorphism (SNP) on FKBP5 methylation, gene expression and post-exercise training adaptations in obesity and metabolic related parameters. Exercise (n = 19) participants underwent 12-weeks of supervised aerobic and resistance training while controls (n = 12) continued their usual behaviours. FKBP5 methylation was measured in GSAT and ASAT using pyrosequencing. SNP and gene expression analyses were conducted using quantitative real-time PCR. Exercise training induced FKBP5 hypermethylation at two CpG dinucleotides within intron 7. When stratified based on the rs1360780 SNP, participants with the CT genotype displayed FKBP5 hypermethylation in GSAT (p < 0.05), and ASAT displayed in both CC and CT carriers. CC allele carriers displayed improved cardiorespiratory fitness, insulin sensitivity, gynoid fat mass, and waist circumference (p < 0.05) in response to exercise training, and these parameters were attenuated in women with the CT genotype. These findings provide a basis for future studies in larger cohorts, which should assess whether FKBP5 methylation and/or genetic variants such as the rs1360780 SNP could have a significant impact on responsiveness to exercise interventions., (© 2022. The Author(s).)

Chronic multisite musculoskeletal pain (CMP) is common and highly morbid. However, vulnerability factors for CMP are poorly understood. Previous studies have independently shown that both small hippocampal brain volume and genetic risk alleles in a key stress system gene, FKBP5, increase vulnerability for chronic pain. However, little is known regarding the relationship between these factors and CMP. Here we tested the hypothesis that both small hippocampal brain volume and FKBP5 genetic risk, assessed using the tagging risk variant, FKBP5rs3800373, increase vulnerability for CMP. We used participant data from 36,822 individuals with available genetic, neuroimaging, and chronic pain data in the UK Biobank study. Although no main effects were observed, the interaction between FKBP5 genetic risk and right hippocampal volume was associated with CMP severity (β = -0.020, p raw  = 0.002, p adj  = 0.01). In secondary analyses, severity of childhood trauma further moderated the relationship between FKBP5 genetic risk, right hippocampal brain volume, and CMP (β = -0.081, p = 0.016). This study provides novel evidence that both FKBP5 genetic risk and childhood trauma moderate the relationship between right hippocampal brain volume and CMP. The data increases our understanding of vulnerability factors for CMP and builds a foundation for further work assessing causal relationships that might drive CMP development., (© 2022. The Author(s).)

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.

Signal peptide peptidase (SPP) is an endoplasmic reticulum (ER)-resident aspartyl protease mediating intramembrane cleavage of type II transmembrane proteins. Increasing evidence has supported the role of SPP in ER-associated protein degradation. In the present study, we show that SPP expression is highly induced in human lung and breast cancers and correlated with disease outcome. Stable depletion of SPP expression in lung and breast cancer cell lines significantly reduced cell growth and migration/invasion abilities. Quantitative analysis of the proteomic changes of microsomal proteins in lung cancer cells by the stable isotope labeling with amino acids in cell culture (SILAC) approach revealed that the level of FKBP8, an endogenous inhibitor of mTOR, was significantly increased following SPP depletion. Co-immunoprecipitation assay and confocal immunofluorescence demonstrated that SPP interacted and colocalized with FKBP8 in ER, supporting that FKBP8 is a protein substrate of SPP. Cycloheximide chase and proteasome inhibition experiments revealed that SPP-mediated proteolysis facilitated FKBP8 protein degradation in cytosol. Further experiment demonstrated that the levels of phosphorylation in mTOR and its downstream effectors, S6K and 4E-BP1, were significantly lower in SPP-depleted cells. The reduced mTOR signaling and decreases of growth and migration/invasion abilities induced by SPP depletion in cancer cells could be reversed by FKBP8 downregulation. The implication of FKBP8 in SPP-mediated tumorigenicity was also observed in the xenograft model. Together, these findings disclose that SPP promotes tumor progression, at least in part, via facilitating the degradation of FKBP8 to enhance mTOR signaling.

Prenatal distress is associated with adverse outcomes in affected offspring. Alterations in placental glucocorticoid signalling and subsequent foetal overexposure to glucocorticoids have been implicated as an underlying mechanism. Infant sex is emerging as an important factor in disease susceptibility. This study aimed to examine the effects of maternal distress across pregnancy on birth outcomes and placental glucocorticoid genes in a sex-dependent manner. Participants completed psychological distress questionnaires throughout pregnancy. Placental HSD11B2, NR3C1 and FKBP51 were analysed by real time PCR and cortisol was measured in new-born hair. Second trimester stress was negatively correlated with birthweight in males and positively correlated with placental NR3C1 mRNA in females. Second trimester anxiety was negatively correlated with birthweight and placental FKBP51 mRNA in females. In mediation analysis, placental FKBP51 mRNA expression was found to mediate the link between prenatal anxiety and birthweight. New-born cortisol was negatively correlated with second trimester anxiety and positively correlated with female placental FKBP51 mRNA levels. Again, FKBP51 mRNA was found to mediate the link between anxiety and new-born cortisol. These results highlight a role for FKBP51 in the placental response to prenatal distress in females. The precise role that placental FKBP51 has in foetal and infant development has not been extensively studied and warrants further investigations.

Antenatal maternal depressive symptoms influence fetal brain development and increase the risk for depression in offspring. Such vulnerability is often moderated by the offspring's genetic variants. This study aimed to examine whether FKBP5, a key regulator of the hypothalamic-pituitary-adrenal (HPA) axis, moderates the association between antenatal maternal depressive symptoms and in utero brain development, using an Asian cohort with 161 mother-offspring dyads. Antenatal maternal depressive symptoms were measured using the Edinburgh Postnatal Depression Scale (EPDS) during the second trimester of pregnancy. Neonatal structural brain images were acquired using magnetic resonance imaging (MRI) shortly after birth. Maternal and neonatal FKBP5 gene was genotyped using Illumina OmniExpress arrays. A gene set-based mixed effect model for gene-environment interaction (MixGE) was used to examine interactive effects between neonatal genetic variants of FKBP5 and antenatal maternal depressive symptoms on neonatal amygdala and hippocampal volumes, and cortical thickness. Our study revealed that genetic variants in neonatal FKBP5 moderate the association between antenatal maternal depressive symptoms and right hippocampal volume but only show a trend for such moderation on amygdala volumes and cortical thickness. Our findings are the first to reveal that the association between maternal depressive symptoms and in utero neurodevelopment of specific brain regions is modified through complex genetic variation in neonatal FKBP5. Our results suggest that an increased risk for depression may be transmitted from mother to child during fetal life and that the effect is dependent upon neonatal FKBP5 genotype.

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Using oral contraceptives has been implicated in the aetiology of stress-related disorders like depression. Here, we followed the hypothesis that oral contraceptives deregulate the HPA-axis by elevating circulating cortisol levels. We report for a sample of 233 pre-menopausal women increased circulating cortisol levels in those using oral contraceptives. For women taking oral contraceptives, we observed alterations in circulating phospholipid levels and elevated triglycerides and found evidence for increased glucocorticoid signalling as the transcript levels of the glucocorticoid-regulated genes DDIT4 and FKBP5 were increased in whole blood. The effects were statistically mediated by cortisol. The associations of oral contraceptives with higher FKBP5 mRNA and altered phospholipid levels were modified by rs1360780, a genetic variance implicated in psychiatric diseases. Accordingly, the methylation pattern of FKBP5 intron 7 was altered in women taking oral contraceptives depending on the rs1360780 genotype. Moreover, oral contraceptives modified the association of circulating cortisol with depressive symptoms, potentially explaining conflicting results in the literature. Finally, women taking oral contraceptives displayed smaller hippocampal volumes than non-using women. In conclusion, the integrative analyses of different types of physiological data provided converging evidence indicating that oral contraceptives may cause effects analogous to chronic psychological stressors regarding the regulation of the HPA axis.

Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

Aberrant restoration of AR activity is linked with prostate tumor growth, therapeutic failures and development of castrate-resistant prostate cancer. Understanding the processes leading to AR-reactivation should provide the foundation for novel avenues of drug discovery. A differential gene expression study was conducted using biopsies from CaP and BPH patients to identify the components putatively responsible for reinstating AR activity in CaP. From the set of genes upregulated in CaP, FKBP52, an AR co-chaperone, was selected for further analysis. Expression of FKBP52 was positively correlated with that of c-Myc. The functional cross-talk between c-Myc and FKBP52 was established using c-Myc specific-siRNA to LNCaP cells that resulted in reduction of FKBP52. A non-canonical E-box sequence housing a putative c-Myc binding site was detected on the FKBP4 promoter using in silico search. LNCaP cells transfected with the FKBP52 promoter cloned in pGL3 basic showed increased luciferase activity which declined considerably when the promoter-construct was co-transfected with c-Myc specific-siRNA. ChIP-PCR confirmed the binding of c-Myc with the conserved E-box located in the FKBP52 promoter. c-Myc downregulation concomitantly affected expression of FGF8. Since expression of FGF8 is controlled by AR, our study unveiled a novel functional axis between c-Myc, AR and FGF8 operating through FKBP52.

The nucleoplasmin family of histone chaperones is identified by a pentamer-forming domain and multiple acidic tracts that mediate histone binding and chaperone activity. Within this family, a novel domain organization was recently discovered that consists of an N-terminal nucleoplasmin-like (NPL) domain and a C-terminal FKBP peptidyl-proline isomerase domain. Saccharomyces cerevisiae Fpr4 is one such protein. Here we report that in addition to its known histone prolyl isomerase activities, the Fpr4 FKBP domain binds to nucleosomes and nucleosome arrays in vitro. This ability is mediated by a collection of basic patches that enable the enzyme to stably associate with linker DNA. The interaction of the Fpr4 FKBP with recombinant chromatin complexes condenses nucleosome arrays independently of its catalytic activity. Based on phylogenetic comparisons we propose that the chromatin binding ability of 'basic' FKBPs is shared amongst related orthologues present in fungi, plants, and insects. Thus, a subclass of FKBP prolyl isomerase enzymes is recruited to linker regions of chromatin.

Paget's disease of bone (PDB) is a common metabolic bone disease that is characterized by aberrant focal bone remodeling, which is caused by excessive osteoclastic bone resorption followed by disorganized osteoblastic bone formation. Genetic factors are a critical determinant of PDB pathogenesis, and several susceptibility genes and loci have been reported, including SQSTM1, TNFSF11A, TNFRSF11B, VCP, OPTN, CSF1 and DCSTAMP. Herein, we report a case of Chinese familial PDB without mutations in known genes and identify a novel c.163G>C (p.Val55Leu) mutation in FKBP5 (encodes FK506-binding protein 51, FKBP51) associated with PDB using whole-exome sequencing. Mutant FKBP51 enhanced the Akt phosphorylation and kinase activity in cells. A study of osteoclast function using FKBP51 V55L KI transgenic mice proved that osteoclast precursors from FKBP51 V55L mice were hyperresponsive to RANKL, and osteoclasts derived from FKBP51 V55L mice displayed more intensive bone resorbing activity than did FKBP51 WT controls. The osteoclast-specific molecules tartrate-resistant acid phosphatase, osteoclast-associated receptor and transcription factor NFATC1 were increased in bone marrow-derived monocyte/macrophage cells (BMMs) from FKBP51 V55L mice during osteoclast differentiation. However, c-fos expression showed no significant difference in the wild-type and mutant groups. Akt phosphorylation in FKBP51 V55L BMMs was elevated in response to RANKL. In contrast, IκB degradation, ERK phosphorylation and LC3II expression showed no difference in wild-type and mutant BMMs. Micro-CT analysis revealed an intensive trabecular bone resorption pattern in FKBP51 V55L mice, and suspicious osteolytic bone lesions were noted in three-dimensional reconstruction of distal femurs from mutant mice. These results demonstrate that the mutant FKBP51 V55L promotes osteoclastogenesis and function, which could subsequently participate in PDB development.

Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and -wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde-derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.

Patients with Cushing's Syndrome (CS) in remission were used as a model to test the hypothesis that long-standing excessive cortisol exposure induces changes in DNA methylation that are associated with persisting neuropsychological consequences. Genome-wide DNA methylation was assessed in 48 women with CS in long-term remission (cases) and 16 controls matched for age, gender and education. The Fatigue impact scale and the comprehensive psychopathological rating scale were used to evaluate fatigue, depression and anxiety. Cases had lower average global DNA methylation than controls (81.2% vs 82.7%; p = 0.002). Four hundred and sixty-one differentially methylated regions, containing 3,246 probes mapping to 337 genes were identified. After adjustment for age and smoking, 731 probes in 236 genes were associated with psychopathology (fatigue, depression and/or anxiety). Twenty-four gene ontology terms were associated with psychopathology; terms related to retinoic acid receptor signalling were the most common (adjusted p = 0.0007). One gene in particular, COL11A2, was associated with fatigue following a false discovery rate correction. Our findings indicate that hypomethylation of FKBP5 and retinoic acid receptor related genes serve a potential mechanistic explanation for long-lasting GC-induced psychopathology.

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a β-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins.

FK506-binding protein 51 (FKBP51) regulates the activity of the glucocorticoid receptor (GR), and is therefore a key mediator of the biological actions of glucocorticoids. However, the understanding of the molecular mechanisms that govern its activity remains limited. Here, we uncover a novel regulatory switch for GR activity by the post-translational modification of FKBP51 with small ubiquitin-like modifier (SUMO). The major SUMO-attachment site, lysine 422, is required for FKBP51-mediated inhibition of GR activity in hippocampal neuronal cells. Importantly, impairment of SUMO conjugation to FKBP51 impacts on GR-dependent neuronal signaling and differentiation. We demonstrate that SUMO conjugation to FKBP51 is enhanced by the E3 ligase PIAS4 and by environmental stresses such as heat shock, which impact on GR-dependent transcription. SUMO conjugation to FKBP51 regulates GR hormone-binding affinity and nuclear translocation by promoting FKBP51 interaction within the GR complex. SUMOylation-deficient FKBP51 fails to interact with Hsp90 and GR thus facilitating the recruitment of the closely related protein, FKBP52, which enhances GR transcriptional activity. Moreover, we show that the modification of FKBP51 with SUMO modulates its binding to Hsp90. Our data establish SUMO conjugation as a novel regulatory mechanism in the Hsp90 cochaperone activity of FKBP51 with a functional impact on GR signaling in a neuronal context.

Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu's H < -20, iHS > 2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

Antidepressants increase hippocampal neurogenesis by activating the glucocorticoid receptor (GR), but excessive GR activation impairs hippocampal neurogenesis, suggesting that normal GR function is crucial for hippocampal neurogenesis. Baicalin was reported to regulate the expression of GR and facilitate hippocampal neurogenesis, but the underlying molecular mechanisms are still unknown. In this study, we used the chronic corticosterone (CORT)-induced mouse model of anxiety/depression to assess antidepressant-like effects of baicalin and illuminate possible molecular mechanisms by which baicalin affects GR-mediated hippocampal neurogenesis. We found that oral administration of baicalin (40, 80 or 160 mg/kg) for 4 weeks alleviated several chronic CORT-induced anxiety/depression-like behaviors. Baicalin also increased Ki-67- and DCX-positive cells to restore chronic CORT-induced suppression of hippocampal neurogenesis. Moreover, baicalin normalized the chronic CORT-induced decrease in GR protein levels, the increase in GR nuclear translocation and the increase in GR phosphorylation at Ser203 and Ser211. Finally, chronic CORT exposure increased the level of FK506-binding protein 51 (FKBP5) and of phosphorylated serum- and glucocorticoid-inducible kinase 1 (SGK1) at Ser422 and Thr256, whereas baicalin normalized these changes. Together, our findings suggest that baicalin improves anxiety/depression-like behaviors and promotes hippocampal neurogenesis. We propose that baicalin may normalize GR function through SGK1- and FKBP5-mediated GR phosphorylation.

Calstabin2, also named FK506 binding protein 12.6 (FKBP12.6), is a subunit of ryanodine receptor subtype 2 (RyR2) macromolecular complex, which is an intracellular calcium channel and abundant in the brain. Previous studies identified a role of leaky neuronal RyR2 in posttraumatic stress disorder (PTSD). However, the functional role of Calstabin2 in the cognitive function remains unclear. Herein, we used a mouse model of genetic deletion of Calstabin2 to investigate the function of Calstabin2 in cognitive dysfunction. We found that Calstabin2 knockout (KO) mice showed significantly reduced performance in Morris Water Maze (MWM), long-term memory (LTM) contextual fear testing, and rotarod test when compared to wild type (WT) littermates. Indeed, genetic deletion of Calstabin2 reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection, increased membrane excitability, and induced RyR2 leak. Finally, we demonstrated that the increase in cytoplasmic calcium activated Ca(2+) dependent potassium currents and led to neuronal apoptosis in KO hippocampal neurons. Thus, these results suggest that neuronal RyR2 Ca(2+) leak due to Calstabin2 deletion contributes to learning deficiency and memory impairment.

Posttraumatic stress disorder (PTSD) is both a prevalent and debilitating trauma-related disorder associated with dysregulated fear learning at the core of many of its signs and symptoms. Improvements in the currently available psychological and pharmacological treatments are needed in order to improve PTSD treatment outcomes and to prevent symptom relapse. In the present study, we used a putative animal model of PTSD that included presentation of immobilization stress (IMO) followed by fear conditioning (FC) a week later. We then investigated the acute effects of GR receptor activation on the extinction (EXT) of conditioned freezing, using dexamethasone administered systemically which is known to result in suppression of the HPA axis. In our previous work, IMO followed by tone-shock-mediated FC was associated with impaired fear EXT. In this study, we administered dexamethasone 4 h before EXT training and then examined EXT retention (RET) 24 h later to determine whether dexamethasone suppression rescued EXT deficits. Dexamethasone treatment produced dose-dependent enhancement of both EXT and RET. Dexamethasone was also associated with reduced amygdala Fkbp5 mRNA expression following EXT and after RET. Moreover, DNA methylation of the Fkbp5 gene occurred in a dose-dependent and time course-dependent manner within the amygdala. Additionally, we found dynamic changes in epigenetic regulation, including Dnmt and Tet gene pathways, as a function of both fear EXT and dexamethasone suppression of the HPA axis. Together, these data suggest that dexamethasone may serve to enhance EXT by altering Fkbp5-mediated glucocorticoid sensitivity via epigenetic regulation of Fkbp5 expression.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).

Detection of protein-protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBPF36V and hetero-interaction of FKBP and FRBT2098L, as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.

Molecular abnormalities within the glucocorticoid receptor (GR) stress signaling pathway may confer, or reflect, susceptibility to stress in schizophrenia and bipolar disorder, but the extent of such abnormalities in the brain is not known. Using RNA-Seq and qPCR in two postmortem cohorts totaling 55 schizophrenia, 34 bipolar disorder and 55 control individuals, we identified increased FKBP5 and PTGES3 mRNA expression, and decreased BAG1 mRNA expression, in the prefrontal cortex in schizophrenia cases relative to controls (68.0% [p < 0.001], 26.0% [p < 0.01] and 12.1% [p < 0.05] respectively). We also observed increased FKBP5 and decreased BAG1 mRNA expression in bipolar disorder (47.5% [p < 0.05] and 14.9% [p < 0.005]). There were no diagnostic differences in steady-state FKBP51 protein levels, nor in HSPA1A, HSP90AA1, DNAJB1 or HSPB1 mRNA levels. GR, co-factor and chaperone mRNA levels were strongly correlated. These results reveal coordinated cortical dysregulation of FKBP5, PTGES3, BAG1 and GR genes within the glucocorticoid signaling pathway in psychotic illness.

FKBP38 regulates apoptosis through unique interactions with multiple regulators including Bcl-2. Interestingly, the peptidylprolyl isomerase activity of FKBP38 is only detectable when it binds to calcium-saturated calmodulin (CaM/Ca(2+)). This, in turn, permits the formation of a complex with Bcl-2. FKBP38 thereby provides an important link between isomerase activity and apoptotic pathways. Here, we show that the N-terminal extension (residues 1-32) preceding the catalytic domain of FKBP38 has an autoinhibitory activity. The core isomerase activity of FKBP38 is inhibited by transient interactions involving the flexible N-terminal extension that precedes the catalytic domain. Notably, CaM/Ca(2+) binds to this N-terminal extension and thereby releases the autoinhibitory contacts between the N-terminal extension and the catalytic domain, thus potentiating the isomerase activity of FKBP38. Our data demonstrate how CaM/Ca(2+) modulates the catalytic activity of FKBP38.

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Various clinical studies have identified FK506-binding protein 51 (FKBP51) as a target gene involved in the development of psychiatric disorders such as depression. Furthermore, FKBP51 has been shown to affect glucocorticoid receptor signaling by sensitivity modulation and it is implicated in stress reactivity as well as in molecular mechanisms of stress vulnerability and resilience. We investigated the physiological, behavioral, and neuroendocrine parameters in an established chronic stress model both directly after stress and after a recovery period of 3 weeks and also studied the efficacy of paroxetine in this model. We then examined FKBP51 mRNA levels in the dorsal and ventral part of the hippocampus and correlated the expression to behavioral and endocrine parameters. We show robust chronic stress effects in physiological, behavioral, and neuroendocrine parameters, which were only slightly affected by paroxetine treatment. On the contrary, paroxetine led to a disruption of the neuroendocrine system. FKBP51 expression was significantly increased directly after the stress period and correlated with behavioral and neuroendocrine parameters. Taken together, we were able to further elucidate the role of FKBP51 in the mechanisms of stress resilience and vulnerability, especially with respect to behavioral and neuroendocrine parameters. These findings strongly support the concept of FKBP51 as a marker for glucocorticoid receptor sensitivity and its involvement in the development of psychiatric disorders.

Although gene expression profiles in peripheral blood in major depression are not likely to identify genes directly involved in the pathomechanism of affective disorders, they may serve as biomarkers for this disorder. As previous studies using baseline gene expression profiles have provided mixed results, our approach was to use an in vivo dexamethasone challenge test and to compare glucocorticoid receptor (GR)-mediated changes in gene expression between depressed patients and healthy controls. Whole genome gene expression data (baseline and following GR-stimulation with 1.5 mg dexamethasone p.o.) from two independent cohorts were analyzed to identify gene expression pattern that would predict case and control status using a training (N=18 cases/18 controls) and a test cohort (N=11/13). Dexamethasone led to reproducible regulation of 2670 genes in controls and 1151 transcripts in cases. Several genes, including FKBP5 and DUSP1, previously associated with the pathophysiology of major depression, were found to be reliable markers of GR-activation. Using random forest analyses for classification, GR-stimulated gene expression outperformed baseline gene expression as a classifier for case and control status with a correct classification of 79.1 vs 41.6% in the test cohort. GR-stimulated gene expression performed best in dexamethasone non-suppressor patients (88.7% correctly classified with 100% sensitivity), but also correctly classified 77.3% of the suppressor patients (76.7% sensitivity), when using a refined set of 19 genes. Our study suggests that in vivo stimulated gene expression in peripheral blood cells could be a promising molecular marker of altered GR-functioning, an important component of the underlying pathology, in patients suffering from depressive episodes.

During apoptosis, mitochondria lose their membrane potential and undergo fragmentation around the time of release of cytochrome c. Apoptotic fission is at least in part sustained by the translocation of dynamin-related protein 1 (Drp1), normally located in the cytosol, to mitochondria. This process depends on dephosphorylation of Drp1 by the phosphatase calcineurin. Here, we report the identification of a novel inhibitor of this process. A polypeptide (PPD1) from the immunophilin FKBP52 inhibits calcineurin activation triggered by mitochondrial dysfunction. PPD1 blocks Drp1 translocation to mitochondria and fragmentation of the organelle. PPD1 delays apoptosis by intrinsic stimuli by preventing fragmentation and release of cytochrome c. Cells expressing PPD1 display enhanced clonogenic ability after exposure to staurosporine. A genetic analysis revealed that the activity of PPD1 is independent of the BH3-only protein BAD, another target of calcineurin during apoptosis, and is not additive to inhibition of Drp1. Thus, PPD1 is a novel inhibitor of apoptosis that elucidates the function of calcineurin-dependent mitochondrial fragmentation in the amplification of cell death.

Prostate cancer (PCa) growth is dependent on androgens and on the androgen receptor (AR), which acts by modulating gene transcription. Tetratricopeptide repeat (TPR) proteins (FKBP52, FKBP51 and Cyp40) interact with AR in PCa cells, suggesting roles in AR-mediated gene transcription and cell growth. We report here that FKBP51 and Cyp40, but not FKBP52, are significantly elevated in PCa tissues and in androgen-dependent (AD) and androgen-independent (AI) cell lines. Overexpression of FKBP51 in AD LNCaP cells increased AR transcriptional activity in the presence and absence of androgen, whereas siRNA knockdown of FKBP51 dramatically decreased AD gene transcription and proliferation. Knockdown of Cyp40 also inhibited androgen-mediated transcription and growth in LNCaP cells. However, disruption of FKBP51 and Cyp40 in AI C4-2 cells caused only a small reduction in proliferation, indicating that Cyp40 and FKBP51 predominantly regulate AD cell proliferation. Under knockdown conditions, the inhibitory effects of TPR ligands, cyclosporine A (CsA) and FK506, on AR activity were not observed, indicating that Cyp40 and FKBP51 are the targets of CsA and FK506, respectively. Our findings show that FKBP51 and Cyp40 are positive regulators of AR that can be selectively targeted by CsA and FK506 to achieve inhibition of androgen-induced cell proliferation. These proteins and their cognate ligands thus provide new strategies in the treatment of PCa.

Chromatin structure is important for controlling gene expression, but mechanisms underlying chromatin remodeling are not fully understood. Here we report that an FKBP (FK506 binding protein) type immunophilin, AtFKBP53, possesses histone chaperone activity and is required for repressing ribosomal gene expression in Arabidopsis. The AtFKBP53 protein is a multidomain FKBP with a typical peptidylprolyl isomerase (PPIase) domain and several highly charged domains. Using nucleosome assembly assays, we showed that AtFKBP53 has histone chaperone activity and the charged acidic domains are sufficient for the activity. We show that AtFKBP53 interacts with histone H3 through the acidic domains, whereas the PPIase domain is dispensable for histone chaperone activity or histone binding. Ribosomal RNA gene (18S rDNA) is overexpressed when AtFKBP53 activity is reduced or eliminated in Arabidopsis plants. Chromatin immunoprecipitation assay showed that AtFKBP53 is associated with the 18S rDNA gene chromatin, implicating that AtFKBP53 represses rRNA genes at the chromatin level. This study identifies a new histone chaperone in plants that functions in chromatin remodeling and regulation of transcription.

FK506-binding protein 51 (FKBP51) is an immunophilin with isomerase activity, which performs important biological functions in the cell. It has recently been involved in the apoptosis resistance of malignant melanoma. The aim of this study was to investigate the possible role of FKBP51 in the control of response to ionizing radiation (Rx) in malignant melanoma. FKBP51-silenced cells showed reduced clonogenic potential after irradiation compared with non-silenced cells. After Rx, we observed apoptosis in FKBP51-silenced cells and autophagy in non-silenced cells. The FKBP51-controlled radioresistance mechanism involves NF-kappaB. FKBP51 was required for the activation of Rx-induced NF-kappaB, which in turn inhibited apoptosis by stimulating X-linked inhibitor of apoptosis protein and promoting authophagy-mediated Bax degradation. Using a tumor-xenograft mouse model, the in vivo pretreatment of tumors with FKBP51-siRNA provoked massive apoptosis after irradiation. Immunohistochemical analysis of 10 normal skin samples and 80 malignant cutaneous melanomas showed that FKBP51 is a marker of melanocyte malignancy, correlating with vertical growth phase and lesion thickness. Finally, we provide evidence that FKBP51 targeting radiosensitizes cancer stem/initiating cells. In conclusion, our study identifies a possible molecular target for radiosensitizing therapeutic strategies against malignant melanoma.

The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.

Aim: The downregulation of phospholamban (PLB) and FKBP12.6 as a result of beta- receptor activation is involved in the pathway(s) of congestive heart failure. We hypothesized that the endothelin (ET)-1 system may link to downregulated PLB and FKBP12.6., Methods: Rats were subjected to ischemia/reperfusion (I/R) to cause heart failure (HF). 1 mg/kg isoproterenol (ISO) was injected subcutaneously (sc) for 10 d to worsen HF. 30 mg/kg CPU0213 (sc), a dual ET receptor (ETAR/ETBR) antagonist was given from d 6 to d 10. On d 11, cardiac function was assessed together with the determination of mRNA levels of ryanodine receptor 2, calstabin-2 (FKBP12.6), PLB, and sarcoplasmic reticulum Ca2+-ATPase. Isolated adult rat ventricular myocytes were incubated with ISO at 1X10(-6) mol/L to set up an in vitro model of HF. Propranolol (PRO), CPU0213, and darusentan (DAR, an ETAR antagonist) were incubated with cardiomyocytes at 1X10(-5) mol/L or 1X10(-6) mol/L in the presence of ISO (1X10(-6) mol/L). Immunocytochemistry and Western blotting were applied for measuring the protein levels of PLB and FKBP12.6., Results: The worsened hemodynamics produced by I/R were exacerbated by ISO pretreatment. The significant downregulation of the gene expression of PLB and FKBP12.6 and worsened cardiac function by ISO were reversed by CPU0213. In vitro ISO 1X10(-6) mol/L produced a sharp decline of PLB and FKBP12.6 proteins relative to the control. The downregulation of the protein expression was significantly reversed by the ET receptor antagonist CPU0213 or DAR, comparable to that achieved by PRO., Conclusion: This study demonstrates a role of ET in mediating the downregulation of the cardiac Ca2+-handling protein by ISO.

Aim: The occurrence of ventricular fibrillation (VF) is dependent on the deterioration of channelopathy in the myocardium. It is interesting to investigate molecular changes in relation to abrupt appearance of VF on reperfusion. We aimed to study whether changes in the expression of FKBP12.6 and SERCA2a and the endothelin (ET) system on reperfusion against ischemia were related to the rapid occurrence of VF and whether CPU86017, a class III antiarrhythmic agent which blocks I(Kr), I(Ks), and I(Ca.L), suppressed VF by correcting the molecular changes on reperfusion., Methods: Cardiomyopathy (CM) was produced by 0.4 mg/kg sc L-thyroxin for 10 d in rats, and subjected to 10 min coronary artery ligation/reperfusion on d 11. Expressions of the Ca2+ handling and ET system and calcium transients were conducted and CPU86017 was injected (4 mg/kg, sc) on d 6-10., Results: A high incidence of VF was found on reperfusion of the rat CM hearts, but there was no VF before reperfusion. The elevation of diastolic calcium was significant in the CM myocytes and exhibited abnormality of the Ca2+ handling system. The rapid downregulation of mRNA and the protein expression of FKBP12.6 and SERCA2a were found on reperfusion in association with the upregulation of the expression of the endothelin-converting enzyme (ECE) and protein kinase A (PKA), in contrast, no change in the ryanodine type 2 receptor (RyR2), phospholamban (PLB), endothelin A receptor (ETAR), and iNOS was found. CPU86017 removed these changes and suppressed VF., Conclusion: Abrupt changes in the expression of FKBP12.6, SERCA2a, PKA, and ECE on reperfusion against ischemia, which are responsible for the rapid occurrence of VF, have been observed. These changes are effectively prevented by CPU86017.

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.