Cannabidiol alleviates neuroinflammation and attenuates neuropathic pain via targeting FKBP5.

Microglia is a heterogeneous population that mediates neuroinflammation in the central nervous system (CNS) and plays a crucial role in developing neuropathic pain. FKBP5 facilitates the assembly of the IκB kinase (IKK) complex for the activation of NF-κB, which arises as a novel target for treating neuropathic pain. In this study, cannabidiol (CBD), a main active component of Cannabis, was identified as an antagonist of FKBP5. In vitro protein intrinsic fluorescence titration showed that CBD directly bound to FKBP5. Cellular thermal shift assay (CETSA) indicated that CBD binding increased the FKBP5 stability, which implies that FKBP5 is the endogenous target of CBD. CBD was found to inhibit the assembly of the IKK complex and the activation of NF-κB, therefore blocking LPS-induced NF-κB downstream pro-inflammatory factors NO, IL-1ß, IL-6 and TNF-α. Stern-Volmer analysis and protein thermal shift assay revealed that tyrosine 113 (Y113) of FKBP5 was critical for FKBP5 interacting with CBD, which is consistent with in silico molecular docking simulation. FKBP5 Y113 mutation (Y113A) alleviated the effect of CBD inhibiting LPS-induced pro-inflammatory factors overproduction. Furthermore, systemic administration of CBD inhibited chronic constriction injury (CCI)-induced microglia activation and FKBP5 overexpression in lumbar spinal cord dorsal horn. These data imply that FKBP5 is an endogenous target of CBD.

Inhibition of FKBP51 induces stress resilience and alters hippocampal neurogenesis.

Stress-related psychiatric disorders such as depression are among the leading causes of morbidity and mortality. Considering that many individuals fail to respond to currently available antidepressant drugs, there is a need for antidepressants with novel mechanisms. Polymorphisms in the gene encoding FK506-binding protein 51 (FKBP51), a co-chaperone of the glucocorticoid receptor, have been linked to susceptibility to stress-related psychiatric disorders. Whether this protein can be targeted for their treatment remains largely unexplored. The aim of this work was to investigate whether inhibition of FKBP51 with SAFit2, a novel selective inhibitor, promotes hippocampal neuron outgrowth and neurogenesis in vitro and stress resilience in vivo in a mouse model of chronic psychosocial stress. Primary hippocampal neuronal cultures or hippocampal neural progenitor cells (NPCs) were treated with SAFit2 and neuronal differentiation and cell proliferation were analyzed. Male C57BL/6 mice were administered SAFit2 while concurrently undergoing a chronic stress paradigm comprising of intermittent social defeat and overcrowding, and anxiety and depressive -related behaviors were evaluated. SAFit2 increased neurite outgrowth and number of branch points to a greater extent than brain derived neurotrophic factor (BDNF) in primary hippocampal neuronal cultures. SAFit2 increased hippocampal NPC neurogenesis and increased neurite complexity and length of these differentiated neurons. In vivo, chronic SAFit2 administration prevented stress-induced social avoidance, decreased anxiety in the novelty-induced hypophagia test, and prevented stress-induced anxiety in the open field but did not alter adult hippocampal neurogenesis in stressed animals. These data warrant further exploration of inhibition of FKBP51 as a strategy to treat stress-related disorders.

FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin.

FK506-binding protein 51 (encoded by Fkpb51, also known as Fkbp5) has been associated with stress-related mental illness. To investigate its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assisted morphological analysis revealed that male Fkbp51 knock-out (KO) mice possess more elongated dentate gyrus (DG) but shorter hippocampal height in coronal sections when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls and pharmacological manipulation experiments suggest that this may occur through the regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support a role for FKBP51 in the regulation of microtubule-associated protein expression. Furthermore, Fkbp51 KO hippocampi exhibited decreases in ßIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory mechanism of Parkin by FKBP51 and the significance of their interaction on disease onset. KO has more flattened hippocampus using AI-assisted measurement Both pyramidal cell layer (PCL) of CA and granular cell layer (GCL) of DG distinguishable as two layers: deep cell layer and superficial layer. Distinct MAP2 expression between deep and superficial layer between KO and WT, Higher Parkin expression in KO brain Mechanism of FKBP51 inhibition resulting in Parkin, MAP2, Tau, and Tubulin expression differences between KO and WT mice, and resulting neurite outgrowth differences.

Targeting the Glucocorticoid Receptor Reduces Binge-Like Drinking in High Drinking in the Dark (HDID-1) Mice.

BACKGROUND: Chronic alcohol exposure can alter glucocorticoid receptor (GR) function in some brain areas that promotes escalated and compulsive-like alcohol intake. GR antagonism can prevent dependence-induced escalation in drinking, but very little is known about the role of GR in regulating high-risk nondependent alcohol intake. Here, we investigate the role of GR in regulating binge-like drinking and aversive responses to alcohol in the High Drinking in the Dark (HDID-1) mice, which have been selectively bred for high blood ethanol (EtOH) concentrations (BECs) in the Drinking in the Dark (DID) test, and in their founder line, the HS/NPT. METHODS: In separate experiments, male and female HDID-1 mice were administered one of several compounds that inhibited GR or its negative regulator, FKBP51 (mifepristone [12.5, 25, 50, 100 mg/kg], CORT113176 [20, 40, 80 mg/kg], and SAFit2 [10, 20, 40 mg/kg]) during a 2-day DID task. EtOH consumption and BECs were measured. EtOH conditioned taste and place aversion (CTA and CPA, respectively) were measured in separate HDID-1 mice after mifepristone administration to assess GR's role in regulating the conditioned aversive effects of EtOH. Lastly, HS/NPT mice were administered CORT113176 during DID to assess whether dissimilar effects from those of HDID-1 would be observed, which could suggest that selective breeding had altered sensitivity to the effects of GR antagonism on binge-like drinking. RESULTS: GR antagonism (with both mifepristone and CORT113176) selectively reduced binge-like EtOH intake and BECs in the HDID-1 mice, while inhibition of FKBP51 did not alter intake or BECs. In contrast, GR antagonism had no effect on EtOH intake or BECs in the HS/NPT mice. Although HDID-1 mice exhibit attenuated EtOH CTA, mifepristone administration did not enhance the aversive effects of EtOH in either a CTA or CPA task. CONCLUSION: These data suggest that the selection process increased sensitivity to GR antagonism on EtOH intake in the HDID-1 mice, and support a role for the GR as a genetic risk factor for high-risk alcohol intake.

The selective FKBP51 inhibitor SAFit2 reduces alcohol consumption and reinstatement of conditioned alcohol effects in mice.

There is still no widely effective pharmacotherapy for alcohol addiction available in the clinic. FK506-binding protein 51 (FKBP51) is a negative regulator of the glucocorticoid receptor signaling pathway that regulates the stress-induced glucocorticoid feedback circuit. Here we asked whether selective inhibitors of FKBP51, exemplified by SAFit2, may serve as a new pharmacological strategy to reduce alcohol consumption and conditioned alcohol effects in a mouse model. We report that a relatively short treatment with SAFit2 (20 mg/kg, ip) reduces ongoing 16 vol% alcohol consumption when administered during free access to alcohol in a two-bottle free-choice test. SAFit2 was also able to reduce alcohol consumption when given during an abstinence period immediately before relapse. In contrast, SAFit2 did not affect alcohol consumption when given during a relapse period after repeated withdrawal from alcohol. SAFit2 (10 and 20 mg/kg, ip) showed no effects when used in an intermittent drinking schedule. When 20 vol% alcohol was only available every other day, SAFit2 had no effect on drinking, no matter whether given during a drinking episode or the day before. SAFit2 (2 and 20 mg/kg, ip) did not affect the expression of an alcohol-induced conditioned place preference (CPP). However, SAFit2 was able to inhibit alcohol-induced reinstatement of an extinguished CPP in a dose-dependent way. Altogether, these data may suggest pharmacological inhibition of FKBP51 as a viable strategy to reduce alcohol seeking and consumption.

Efficient and Orthogonal Transcription Regulation by Chemically Inducible Artificial Transcription Factors.

The DNA-binding specificity of genome editing tools can be applied to gene regulation. Recently, multiple artificial transcription factors (ATFs) were shown to synergistically and efficiently regulate gene expression. Chemically triggered protein associations are useful for functional regulation at specific timings. A combination of several inducible protein association systems could enable the regulation of multiple genes at different loci with independent timing. We applied the FKBP-rapamycin-FRB and GAI-gibberellin-GID systems for gene regulation using multiple TALEs and dCas9. By the combined use of currently available systems, reporter gene assays were performed; the results indicated that gene expression was regulated by rapamycin or gibberellin in the presence of the FRB or GAI effector domains, respectively. Furthermore, the activation of endogenous genes was differentially regulated by the system. This success suggests the usability of the chemically inducible multiple ATFs for the time-dependent regulation of multiple genes, such as the case for cellular phenomena that are dependent on the programmable timing of expression and the differential expression of multiple genes.

Amyloid ß production is regulated by ß2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

Alteration of ryanodine receptor (RyR)-mediated calcium (Ca2+) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the ß-amyloid precursor protein (ßAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid ß (Aß), ß-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aß through a ß2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances ßAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the ß2-adrenergic signaling cascade reduced ßAPP processing and the production of Aß in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.

Molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis on selectivity difference between FKBP51 and FKBP52: Insight into the molecular mechanism of isoform selectivity.

As co-chaperones of the 90-kDa heat shock protein(HSP90), FK506 binding protein 51 (FKBP51) and FK506 binding protein 52 (FKBP52) modulate the maturation of steroid hormone receptor through their specific FK1 domains (FKBP12-like domain 1). The inhibitors targeting FK1 domains are potential therapies for endocrine-related physiological disorders. However, the structural conservation of the FK1 domains between FKBP51 and FKBP52 make it difficult to obtain satisfactory selectivity in FK506-based drug design. Fortunately, a series of iFit ligands synthesized by Hausch et al exhibited excellent selectivity for FKBP51, providing new opportunity for design selective inhibitors. We performed molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis to reveal selective mechanism for the inhibitor iFit4 binding with FKBP51 and FKBP52. The conformational stability evaluation of the "Phe67-in" and "Phe67-out" states implies that FKBP51 and FKBP52 have different preferences for "Phe67-in" and "Phe67-out" states, which we suggest as the determinant factor for the selectivity for FKBP51. The binding free energy calculations demonstrate that nonpolar interaction is favorable for the inhibitors binding, while the polar interaction and entropy contribution are adverse for the inhibitors binding. According to the results from binding free energy decomposition, the electrostatic difference of residue 85 causes the most significant thermodynamics effects on the binding of iFit4 to FKBP51 and FKBP52. Furthermore, the importance of substructure units on iFit4 were further evaluated by unbinding pathway analysis and residue-residue contact analysis between iFit4 and the proteins. The results will provide new clues for the design of selective inhibitors for FKBP51.

Modeling Analysis of Potential Target of Dolastatin 16 by Computational Virtual Screening.

Dolastatin 16 is a cyclic depsipeptide isolated from the marine invertebrates and cyanobacterium Lyngbya majuscula, however, its bioactivity has been a historical question. In this study, peptidyl-prolyl cis-trans isomerase FKBP1A (FKBP12) was predicted as a potential target of dolastatin 16 via PharmMapper as well as verified using chemical-protein interactome (CPI) and molecular docking. FKBP1A has been previously identified as a target for the natural polyketide FK506 (tacrolimus), an immune suppressor inhibiting the rejection of organ transplantation in clinical use. The comparison study via the reverse pharmacophore screening and molecular docking of dolastatin 16 and FK506 indicated the good consistency of analysis with the computational approach. As the results, the lowest binding energy of dolastatin 16-FKBP1A complex was -7.4 kcal/mol and FK506-FKBP1A complex was -8.7 kcal/mol. The ligand dolastatin 16 formed three hydrogen bonds vs. four of FK506, as well as seven hydrophobic interactions vs. six of FK506 within the active site residues. These functional residues are highly repetitive and consistent with previously reported active site of model of FK506-FKBP1A complex, and the pharmacophore model was shown feasibly matching with the molecular feature of dolastatin 16.

Selective inhibitors of the FK506-binding protein 51 by induced fit.

The FK506-binding protein 51 (FKBP51, encoded by the FKBP5 gene) is an established risk factor for stress-related psychiatric disorders such as major depression. Drug discovery for FKBP51 has been hampered by the inability to pharmacologically differentiate against the structurally similar but functional opposing homolog FKBP52, and all known FKBP ligands are unselective. Here, we report the discovery of the potent and highly selective inhibitors of FKBP51, SAFit1 and SAFit2. This new class of ligands achieves selectivity for FKBP51 by an induced-fit mechanism that is much less favorable for FKBP52. By using these ligands, we demonstrate that selective inhibition of FKBP51 enhances neurite elongation in neuronal cultures and improves neuroendocrine feedback and stress-coping behavior in mice. Our findings provide the structural and functional basis for the development of mechanistically new antidepressants.

Pharmacological Inhibition of the Psychiatric Risk Factor FKBP51 Has Anxiolytic Properties.

Anxiety-related psychiatric disorders represent one of the largest health burdens worldwide. Single nucleotide polymorphisms of the FK506 binding protein 51 (FKBP51) gene have been repeatedly associated with anxiety-related disorders and stress sensitivity. Given the intimate relationship of stress and anxiety, we hypothesized that amygdala FKBP51 may mediate anxiety-related behaviors. Mimicking the stress effect by specifically overexpressing FKBP51 in the basolateral amygdala (BLA) or central amygdala resulted in increased anxiety-related behavior, respectively. In contrast, application of a highly selective FKBP51 point mutant antagonist, following FKBP51(mut) BLA-overexpression, reduced the anxiogenic phenotype. We subsequently tested a novel FKBP51 antagonist, SAFit2, in wild-type mice via BLA microinjections, which reduced anxiety-related behavior. Remarkably, the same effect was observed following peripheral administration of SAFit2. To our knowledge, this is the first in vivo study using a specific FKBP51 antagonist, thereby unraveling the role of FKBP51 and its potential as a novel drug target for the improved treatment of anxiety-related disorders.

Computational insights into the suicide inhibition of Plasmodium falciparum Fk506-binding protein 35.

Malaria is a parasite affecting millions of people worldwide. With the risk of malarial resistance reaching catastrophic levels, novel methods into the inhibition of this disease need to be prioritized. The exploitation of active site differences between parasitic and human peptidyl-prolyl cis/trans isomerases can be used for suicide inhibition, effectively poisoning the parasite without affecting the patient. This method of inhibition was explored using Plasmodium falciparum and Homo sapiens Fk506-binding proteins as templates for quantum mechanics/molecular mechanics calculations. Modification of the natural substrate has shown suicide inhibition is a valid approach for novel anti-malarials with little risk for parasitic resistance.

Rational design and asymmetric synthesis of potent and neurotrophic ligands for FK506-binding proteins (FKBPs).

To create highly efficient inhibitors for FK506-binding proteins, a new asymmetric synthesis for pro-(S)-C(5) -branched [4.3.1] aza-amide bicycles was developed. The key step of the synthesis is an HF-driven N-acyliminium cyclization. Functionalization of the C(5)  moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280-fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.

FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells.

Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca(2+) channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca(2+) entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca(2+)](c)), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca(2+) mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn(2+) entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP(3)R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells.

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

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.

MicroRNA-100/99a, deregulated in acute lymphoblastic leukaemia, suppress proliferation and promote apoptosis by regulating the FKBP51 and IGF1R/mTOR signalling pathways.

BACKGROUND: MicroRNAs alter multiple cell processes and thus influence tumour carcinogenesis and progression. MiR-100 and miR-99a have been reported to be aberrantly expressed in acute leukaemia. In this study, we focused on their functions in acute lymphoblastic leukaemia (ALL) and the molecular networks in which they are involved. METHODS: MiR-100 and miR-99a expression levels were measured in acute leukaemia patients by qRT-PCR. Kaplan-Meier analysis and log-rank tests were used to calculate the survival rate. Three human ALL cell lines were studied. Apoptosis and proliferation were analysed using siRNA transfection, western blot and flow cytometry. RESULTS: In vivo, miR-100 and miR-99a were down-regulated in 111 ALL patients, especially in high-risk groups; their expression levels were correlated with the patient's 5-year survival. In vitro, the restoration of miR-100 and miR-99a in ALL cells suppressed cell proliferation and increased dexamethasone-induced cell apoptosis. Ectopic expression of miR-100 and miR-99a targeted FK506-binding protein 51 (FKBP51) and, in turn, influenced glucocorticoid receptor (GR) activity. Meanwhile, miR-100 and miR-99a overexpression inhibited the expression of IGF1R and mTOR and their downstream oncogene MCL1. CONCLUSION: MiR-100 and miR-99a have critical roles in altering cellular processes by targeting both the FKBP51 and IGF1R/mTOR signalling pathways in vitro and might represent a potential novel strategy for ALL treatment.

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells.

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.

Domain structure and denaturation of a dimeric Mip-like peptidyl-prolyl cis-trans isomerase from Escherichia coli.

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

Hypothalamic Fkbp51 is induced by fasting, and elevated hypothalamic expression promotes obese phenotypes.

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.