FKBP12 Research Articles

FKBP4 promotes glycolysis and hepatocellular carcinoma progression via p53/HK2 axis.

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 and18F-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 and18F-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.

Single-Molecule-Based, Label-Free Monitoring of Molecular Glue Efficacies for Promoting Protein-Protein Interactions Using YaxAB Nanopores.

Modulating protein-protein interactions (PPIs) is an attractive strategy in drug discovery. Molecular glues, bifunctional small-molecule drugs that promote PPIs, offer an approach to targeting traditionally undruggable targets. However, the efficient discovery of molecular glues has been hampered by the current limitations of conventional ensemble-averaging-based methods. In this study, we present a YaxAB nanopore for probing the efficacy of molecular glues in inducing PPIs. Using YaxAB nanopores, we demonstrate single-molecule-based, label-free monitoring of protein complex formation between mammalian target of rapamycin (mTOR) and FK506-binding proteins (FKBPs) triggered by the molecular glue, rapamycin. Owing to its wide entrance and adjustable pore size, in combination with potent electro-osmotic flow (EOF), a single funnel-shaped YaxAB nanopore enables the simultaneous detection and single-molecule-level quantification of multiprotein states, including single proteins, binary complexes, and ternary complexes induced by rapamycin. Notably, YaxAB nanopores could sensitively discriminate between the binary complexes or ternary complexes induced by rapamycin and its analogues, despite the subtle size differences of ∼122 or ∼116 Da, respectively. Taken together, our results provide proof-of-concept for single-molecule-based, label-free, and ultrasensitive screening and structure-activity relationship (SAR) analysis of molecular glues, which will contribute to low-cost, highly efficient discovery, and rational design of bifunctional modality of drugs, such as molecular glues.

Leonurine Inhibits Hepatic Lipid Synthesis to Ameliorate NAFLD via the ADRA1a/AMPK/SCD1 Axis.

Leonurine is a natural product unique to the Lamiaceae plant Leonurus japonicus Houtt., and it has attracted attention due to its anti-oxidative stress, anti-apoptosis, anti-fibrosis, and metabolic regulation properties. Also, it plays an important role in the prevention and treatment of nonalcoholic fatty liver disease (NAFLD) through a variety of biological mechanisms, but its mechanism of action remains to be elucidated. Therefore, this study aims to preliminarily explore the mechanisms of action of leonurine in NAFLD. Mice were randomly divided into four groups: the normal control (NC) group, the Model (M) group, the leonurine treatment (LH) group, and the fenofibrate treatment (FB) group. The NAFLD model was induced by a high-fat high-sugar diet (HFHSD) for 12 weeks, and liver pathological changes and biochemical indices were observed after 12 weeks. Transcriptomic analysis results indicated that leonurine intervention reversed the high-fat high-sugar diet-induced changes in lipid metabolism-related genes such as stearoyl-CoA desaturase 1 (Scd1), Spermine Synthase (Sms), AP-1 Transcription Factor Subunit (Fos), Oxysterol Binding Protein Like 5 (Osbpl5), and FK506 binding protein 5 (Fkbp5) in liver tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results suggest that leonurine may exert its lipid-lowering effects through the AMP-activated protein kinase (AMPK) signaling pathway. Liver lipidomic analysis showed that leonurine could alter the abundance of lipid molecules related to fatty acyl (FAs) and glycerophospholipids (GPs) such as TxB3, carnitine C12-OH, carnitine C18:1-OH, and LPC (20:3/0:0). Molecular biology experiments and molecular docking techniques verified that leonurine might improve hepatic lipid metabolism through the alpha-1A adrenergic receptor (ADRA1a)/AMPK/SCD1 axis. In summary, the present study explored the mechanism by which leonurine ameliorated NAFLD by inhibiting hepatic lipid synthesis via the ADRA1a/AMPK/SCD1 axis.

Changes of Ca2+ release through ryanodine receptor 1 in pork under different postmortem chilling rates

The role of postmortem chilling rate in regulating Ca2+ release through ryanodine receptor 1 (RyR1) in pork microstructure and tenderness were evaluated. The pork was allocated to the low chilling rate (LCR, 4.26°C/h), medium chilling rate (MCR, 8.89°C/h), and high chilling rate (HCR, 13.33°C/h) group. Sarcomere super-contraction and fiber damage were obtained in HCR samples, leading to the lowest shear force (P < 0.05). Under HCR, Ca2+ concentration significantly increased in sarcoplasm and decreased in sarcoplasmic reticulum were found at 2-5 h postmortem compared to other groups (P < 0.05). RyR1 and dihydropyridine receptor (DHPR) levels were higher and 12 kDa FK506 binding protein (FKBP12) level was lower in HCR samples at 6 h postmortem (P < 0.05). Therefore, HCR (13.33°C/h) may enhance Ca2+ release by up-regulating RyR1 level with DHPR activation increasing and FKBP12 inhibition decreasing, causing myofibril damage from sarcomere super-contraction to improve pork tenderness at early postmortem.

USP15-Mediated Deubiquitination of FKBP 5 and Activation of the αIIbβ3 Signaling Pathway Regulate Thrombosis in Mice.

Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbβ3 in washed platelets was analyzed using flow cytometry. USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbβ3 in mice. USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbβ3.

Fkbp5 gene deletion: Circadian rhythm profile and brain proteomics in aged mice.

FKBP51, also known as FK506-binding protein 51, is a molecular chaperone and scaffolding protein with significant roles in regulating hormone signaling and responding to stress. Genetic variants in FKBP5, which encodes FKBP51, have been implicated in a growing number of neuropsychiatric disorders, which has spurred efforts to target FKBP51 therapeutically. However, the molecular mechanisms and sub-anatomical regions influenced by FKBP51 in these disorders are not fully understood. In this study, we aimed to examine the impact of Fkbp5 ablation using circadian phenotyping and molecular analyses. Our findings revealed that the lack of FKBP51 did not significantly alter circadian rhythms, as detected by wheel-running activity, but did offer protection against stress-mediated disruptions in rhythmicity in a sex-dependent manner. Protein changes in Fkbp5 KO mice, as measured by histology and proteomics, revealed alterations in a brain region- and sex-dependent manner. Notably, regardless of sex, aged Fkbp5 KOs showed elevated MYCBP2, FBXO45, and SPRYD3 levels, which are associated with neuronal-cell adhesion and synaptic integrity. Additionally, pathways such as serotonin receptor signaling and S100 family signaling were differentially regulated in Fkbp5 KO mice. Weighted protein correlation network analysis identified protein networks linked with synaptic transmission and neuroinflammation. The information generated by this work can be used to better understand the molecular changes in the brain during aging and in the absence of Fkbp5, which has implications for the continued development of FKBP51-focused therapeutics for stress-related disorders.

New Features in Visual Dynamics 3.0.

Visual Dynamics (VD) is a web tool that aims to facilitate the use and application of Molecular Dynamics (MD) executed in Gromacs, allowing users without computational familiarity to run short-time simulations for validation, demonstration, and teaching purposes. It is true that quantum methods are the most accurate. However, there is currently no computational feasibility to carry out the experiments that MD performs. The tool described here has continuously received improvements over the course of the last couple of years. This protocol will describe what is needed to run a simulation in VD with a protein-ligand complex previously prepared in ACPYPE and some general directions on the other simulation models available. For the detailed simulation, the FK506-binding protein from Plasmodium vivax complexed with the inhibitor D5 (PDB ID: 4mgv) will be used, and all files used will be provided. Note that this protocol will tell every option to be used to achieve the same results presented, but these options are not necessarily the only ones available.

Structure-Based Design of Ultrapotent Tricyclic Ligands for FK506-Binding Proteins.

Access to small, rigid, and sp3-rich molecules is a major limitation in the drug discovery for challenging protein targets. FK506-binding proteins hold high potential as drug targets or enablers of molecular glues but are fastidious in the chemotypes accepted as ligands. We here report an enantioselective synthesis of a highly rigidified pipecolate-mimicking tricyclic scaffold that precisely positions functional groups for interacting with FKBPs. This was enabled by a 14-step gram-scale synthesis featuring anodic oxidation, stereospecific vinylation, and N-acyl iminium cyclization. Structure-based optimization resulted in the discovery of FKBP inhibitors with picomolar biochemical and subnanomolar cellular activity that represent the most potent FKBP ligands known to date.

Primary cilia mediate skeletogenic BMP and Hedgehog signaling in heterotopic ossification.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 (ACVR1). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1Q207D, Sox2-Cre; Acvr1R206H/+ FOP mice and in burn tenotomy-treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.

Single-cell sequencing of facial adipose tissue unveils FKBP5 as a therapeutic target for facial infiltrating lipomatosis

BackgroundFacial infiltrating lipomatosis is characterized by excessive growth of adipose tissue. Its etiology is associated with somatic phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) variants, but the specific mechanisms are not yet fully understood.MethodsWe collected facial adipose tissue from both FIL patients and non-FIL individuals, isolated the stromal vascular fraction (SVF) and performed single-cell transcriptome sequencing on these samples.ResultsWe mapped out the cellular landscape within the SVF, with a specific focus on a deeper analysis of fibro-adipogenic precursor cells (FAPs). Our analysis revealed that FAPs from FIL patients (FIL-FAPs) significantly overexpressed FK506 binding protein 51 (FKBP5) compared to FAPs from individuals without FIL. Further experiments indicated that FKBP5 is regulated by the PI3K-AKT signaling pathway. The overactivation of this pathway led to an increase in FKBP5 expression. In vitro experiments demonstrated that FKBP5 promoted adipogenic differentiation of FAPs, a process that could be hindered by FKBP5 knockdown or inhibition. Additionally, in vivo assessments confirmed FKBP5’s role in adipogenesis.ConclusionsThese insights into the pathogenesis of FIL underscore FKBP5 as a promising target for developing non-surgical interventions to manage the excessive adipose tissue growth in FIL.

The target of rapamycin signaling pathway regulates vegetative development, aflatoxin biosynthesis, and pathogenicity in Aspergillus flavus.

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. Despite its significance, the underlying mechanism of the TOR pathway in Aspergillus flavus remains elusive. In this study, we comprehensively analyzed the TOR signaling pathway in A. flavus by identifying and characterizing nine genes that encode distinct components of this pathway. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The TorA kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and responses to rapamycin and cell membrane stress. As a significant downstream effector molecule of the TorA kinase, the Sch9 kinase regulates aflatoxin B1 (AFB1) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S_TKc, S_TK_X domains, and the ATP-binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the TorA kinase, play a significant role in regulating cell wall stress response in A. flavus. Moreover, the members of the TapA-phosphatase complexes, SitA and Ppg1, are important for various biological processes in A. flavus, including vegetative growth, sclerotia formation, AFB1 biosynthesis, and pathogenicity. We also demonstrated that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathways. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin production, and LDs biogenesis. Collectively, our study has provided important insight into the regulatory network of the TOR signaling pathway and has elucidated the underlying molecular mechanisms of aflatoxin biosynthesis in A. flavus.

Genome-Wide Identification and Bioinformatics Analysis of the FK506 Binding Protein Family in Rice.

The FK506 Binding Protein (FKBP), ubiquitously present across diverse species, is characterized by its evolutionarily conserved FK506 binding domain (FKBd). In plants, evidence suggests that this gene family plays integral roles in regulating growth, development, and responses to environmental stresses. Notably, research on the identification and functionality of FKBP genes in rice remains limited. Therefore, this study utilized bioinformatic tools to identify 30 FKBP-encoding genes in rice. It provides a detailed analysis of their chromosomal locations, evolutionary relationships with the Arabidopsis thaliana FKBP family, and gene structures. Further analysis of the promoter elements of these rice FKBP genes revealed a high presence of stress-responsive elements. Quantitative PCR assays under drought and heat stress conditions demonstrated that genes OsFKBP15-2, OsFKBP15-3, OsFKBP16-3, OsFKBP18, and OsFKBP42b are inducible by these adverse conditions. These findings suggest a significant role for the rice FKBP gene family in stress adaptation. This research establishes a critical foundation for deeper explorations of the functional roles of the OsFKBP genes in rice.

1,4-Pyrazolyl-Containing SAFit-Analogues are Selective FKBP51 Inhibitors With Improved Ligand Efficiency and Drug-Like Profile.

The FK506 binding protein 51 (FKBP51) is an appealing drug target due to its role in several diseases such as depression, anxiety, chronic pain and obesity. Towards this, selectivity versus the close homolog FKBP52 is essential. However, currently available FKBP51-selective ligands such as SAFit2 are too large and lack drug-like properties. Here, we present a structure activity relationship (SAR) analysis of the pipecolic ester moiety of SAFit1 and SAFit2, which culminated in the discovery of the 1,4-pyrazolyl derivative 23 d, displaying a binding affinity of 0.077 μM for FKBP51, reduced molecular weight (541.7 g/mol), lower hydrophobicity (cLogP=3.72) and higher ligand efficiency (LE=0.25). Cocrystal structures revealed the importance of the 1,4- and 1,3,4- substitution patterns of the pyrazole ring versus the 1,4,5 arrangement.

A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.

FKBP3, a poor prognostic indicator, promotes the progression of LUAD via regulating ferroptosis and immune infiltration.

Ferroptosis was reported to possess the therapeutic potentials in various human cancers. In the present study, we explored the expression, clinical significance and the molecular mechanism of FK506 binding protein 3 (FKBP3) in the progression of lung adenocarcinoma (LUAD). Cox regression was performed to obtain the prognosis related to differentially expressed genes (DEGs) in LUAD datasets from TCGA. We also downloaded the ferroptosis-related gene datasets from GeneCards. Venn diagram was performed to find the intersecting genes and FKBP3 was selected as the targeted gene by analyzing the diagnostic and prognostic values of Top10 intersecting genes. Moreover, univariate and multivariate analyses were performed to evaluate the association between clinicopathological factors and survival rates. GO/KEGG and GSEA analysis was performed to explore the function of FKBP3 in LUAD progression. Protein-protein interaction (PPI) network was performed via STRING database and the top10 hub genes were selected. Finally, the relationship between FKBP3 and immune infiltration was explored by ssGSEA analysis. Firstly, 184 genes associated with the prognosis of LUAD and ferroptosis were obtained. FKBP3 was found to be significantly associated with a poor overall survival rate of LUAD patients. Immunohistochemical staining results showed that FKBP3 was highly located in cytoplasm and membrane of cells in LUAD tissues. PPI network analysis results showed that HDAC1, YY1, HDAC2, MTOR, PSMA3, PIN1, NCL, C14orf166, PIN4, and LARP6 were the top10 hub genes. Furthermore, spearman analysis results showed that the expression of FKBP3 was positively correlated with the abundance of Th2 cells and T helper cells. High level of FKBP3 was associated with poor prognostic outcomes of LUAD patients, which also inhibited immune infiltration in LUAD tissues. Additionally, FKBP3 was involved in regulating the ferroptosis process in LUAD patients. Thus, FKBP3 possessed the tumor promotion role might be involving in regulating ferroptosis and immune infiltration in LUAD progression.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FK506-binding protein 12 (FKBP12)-rapamycin-binding (FRB) domain of human target of rapamycin kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FRB domain fused to the target proteins with the human FKBP fused to S-phase kinase-associated protein 1 or Cullin 1, subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein dynamin-related protein 5B and E2 transcription factor, a regulator of the G1/S transition, within 2 to 3 h after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

NFAT activation by FKBP52 promotes cancer cell proliferation by suppressing p53.

FK506-binding protein 52 (FKBP52) is a member of the FKBP family of proline isomerases. FKBP52 is up-regulated in various cancers and functions as a positive regulator of steroid hormone receptors. Depletion of FKBP52 is known to inhibit cell proliferation; however, the detailed mechanism remains poorly understood. In this study, we found that FKBP52 depletion decreased MDM2 transcription, leading to stabilization of p53, and suppressed cell proliferation. We identified NFATc1 and NFATc3 as transcription factors that regulate MDM2 We also found that FKBP52 associated with NFATc3 and facilitated its nuclear translocation. In addition, calcineurin, a well-known Ca2+ phosphatase essential for activation of NFAT, plays a role in MDM2 transcription. Supporting this notion, MDM2 expression was found to be regulated by intracellular Ca2+ Taken together, these findings reveal a new role of FKBP52 in promoting cell proliferation via the NFAT-MDM2-p53 axis, and indicate that inhibition of FKBP52 could be a new therapeutic tool to activate p53 and inhibit cell proliferation.

The Role of FKBPs in Complex Disorders: Neuropsychiatric Diseases, Cancer, and Type 2 Diabetes Mellitus.

Stress is a common denominator of complex disorders and the FK-506 binding protein (FKBP)51 plays a central role in stress. Hence, it is not surprising that multiple studies imply the involvement of the FKBP51 protein and/or its coding gene, FKBP5, in complex disorders. This review summarizes such reports concentrating on three disorder clusters-neuropsychiatric, cancer, and type 2 diabetes mellitus (T2DM). We also attempt to point to potential mechanisms suggested to mediate the effect of FKBP5/FKBP51 on these disorders. Neuropsychiatric diseases considered in this paper include (i) Huntington's disease for which increased autophagic cellular clearance mechanisms related to decreased FKBP51 protein levels or activity is discussed, Alzheimer's disease for which increased FKBP51 activity has been shown to induce Tau phosphorylation and aggregation, and Parkinson's disease in the context of which FKBP12 is mentioned; and (ii) mental disorders, for which significant association with the single nucleotide polymorphism (SNP) rs1360780 of FKBP5 intron 7 along with decreased DNA methylation were revealed. Since cancer is a large group of diseases that can start in almost any organ or tissue of the body, FKBP51's role depends on the tissue type and differences among pathways expressed in those tumors. The FKBP51-heat-shock protein-(Hsp)90-p23 super-chaperone complex might function as an oncogene or as a tumor suppressor by downregulating the serine/threonine protein kinase (AKt) pathway. In T2DM, two potential pathways for the involvement of FKBP51 are highlighted as affecting the pathogenesis of the disease-the peroxisome proliferator-activated receptor-γ (PPARγ) and AKt.