Marc B. Cox

FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior.

BACKGROUND The Hsp90 cochaperone FK506 binding protein 5 (FKBP5) is an established regulator of the glucocorticoid receptor (GR), and numerous genetic studies have linked it to stress-related diseases such as major depression or posttraumatic stress disorder. However, translational studies including genetic animal models are lacking. METHODS Mice deficient of FKBP5 were generated and analyzed in comparison with wildtype littermates. They were subjected to several test paradigms characterizing their emotionality, stress reactivity, and coping behavior as well as hypothalamus-pituitary-adrenal axis function and regulation. Moreover, protein expression of GR and FKBP5 was determined in different brain structures 8 days after stress exposure. The combined dexamethasone/corticotropin-releasing hormone test was performed both in mice and healthy human subjects of different FKBP5 genotypes. The GR function was evaluated by reporter gene assays. RESULTS Under basal conditions, deletion of FKBP5 did not change exploratory drive, locomotor activity, anxiety-related behavior, stress-coping, or depression-like behavior. After exposure to different acute stressors of sufficient intensity, however, it led to a more active coping behavior. Moreover, loss of FKBP5 decreased hypothalamus-pituitary-adrenal axis reactivity and GR expression changes in response to stressors. In mice and humans, the FKBP5 genotype also determined the outcome of the dexamethasone/corticotropin-releasing hormone test. CONCLUSIONS This study in mice and humans presents FKBP5 as a decisive factor for the physiological stress response, shaping neuroendocrine reactivity as well as coping behavior. This lends strong support to the concept emerging from human studies of FKBP5 as important factor governing gene-environment interactions relevant for the etiology of affective disorders.

The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules.

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimers disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

FKBP51 and FKBP52 in signaling and disease

FKBP51 and FKBP52 are diverse regulators of steroid hormone receptor signaling, including receptor maturation, hormone binding and nuclear translocation. Although structurally similar, they are functionally divergent, which is largely attributed to differences in the FK1 domain and the proline-rich loop. FKBP51 and FKBP52 have emerged as likely contributors to a variety of hormone-dependent diseases, including stress-related diseases, immune function, reproductive functions and a variety of cancers. In addition, recent studies have implicated FKBP51 and FKBP52 in Alzheimers disease and other protein aggregation disorders. This review summarizes our current understanding of FKBP51 and FKBP52 interactions within the receptor-chaperone complex, their contributions to health and disease, and their potential as therapeutic targets for the treatment of these diseases.

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop.

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90‐binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α‐helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A–TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide‐binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C‐terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.

Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase domain in the regulation of steroid hormone signaling

ABSTRACT Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.

Functional Specificity of Co-Chaperone Interactions with Hsp90 Client Proteins

A wide array of proteins in signal transduction pathways depend on Hsp90 and other chaperone components for functional maturation, regulation, and stability. Among these Hsp90 client proteins are steroid receptors, members from other classes of transcription factors, and representatives of both serine/threonine and tyrosine kinase families. Typically, dynamic complexes form on the client protein, and these consist of Hsp90- plus bound co-chaperones that often have enzymatic activities. In addition to its direct influence on client folding, Hsp90 locally concentrates co-chaperone activity within the client complex, and dynamic exchange of co-chaperones on Hsp90 facilitates sampling of co-chaperone activities that may, or may not, act on the client protein. We are just beginning to understand the nature of biochemical and molecular interactions between co-chaperone and Hsp90-bound client. This review focuses on the differential effects of Hsp90 co-chaperones toward client protein function and on the specificity that allows co-chaperones to discriminate between even closely related clients.

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.

A New Anti-Depressive Strategy for the Elderly: Ablation of FKBP5/FKBP51

The gene FKBP5 codes for FKBP51, a co-chaperone protein of the Hsp90 complex that increases with age. Through its association with Hsp90, FKBP51 regulates the glucocorticoid receptor (GR). Single nucleotide polymorphisms (SNPs) in the FKBP5 gene associate with increased recurrence of depressive episodes, increased susceptibility to post-traumatic stress disorder, bipolar disorder, attempt of suicide, and major depressive disorder in HIV patients. Variation in one of these SNPs correlates with increased levels of FKBP51. FKBP51 is also increased in HIV patients. Moreover, increases in FKBP51 in the amygdala produce an anxiety phenotype in mice. Therefore, we tested the behavioral consequences of FKBP5 deletion in aged mice. Similar to that of naïve animals treated with classical antidepressants FKBP5−/− mice showed antidepressant behavior without affecting cognition and other basic motor functions. Reduced corticosterone levels following stress accompanied these observed effects on depression. Age-dependent anxiety was also modulated by FKBP5 deletion. Therefore, drug discovery efforts focused on depleting FKBP51 levels may yield novel antidepressant therapies.

Regulation of steroid hormone receptor function by the 52-kDa FK506-binding protein (FKBP52)

The large FK506-binding protein FKBP52 has been characterized as an important positive regulator of androgen, glucocorticoid and progesterone receptor signaling pathways. FKBP52 associates with receptor-Hsp90 complexes and is proposed to have roles in both receptor hormone binding and receptor subcellular localization. Data from biochemical and cellular studies have been corroborated in whole animal models as fkbp52-deficient male and female mice display characteristics of androgen, glucocorticoid and/or progesterone insensitivity. FKBP52 receptor specificity and the specific phenotypes displayed by the fkbp52-deficient mice have firmly established FKBP52 as a promising target for the treatment of a variety of hormone-dependent diseases. Recent studies demonstrated that the FKBP52 FK1 domain and the proline-rich loop within this domain are functionally important for FKBP52 regulation of receptor function. Based on these data, efforts are currently underway to target the FKBP52 FK1 domain and the proline-rich loop with small molecule inhibitors.

A four-hour yeast bioassay for the direct measure of estrogenic activity in wastewater without sample extraction, concentration, or sterilization.

The assay described here represents an improved yeast bioassay that provides a rapid yet sensitive screening method for EDCs with very little hands-on time and without the need for sample preparation. Traditional receptor-mediated reporter assays in yeast were performed twelve to twenty four hours after ligand addition, used colorimetric substrates, and, in many cases, required high, non-physiological concentrations of ligand. With the advent of new chemiluminescent substrates a ligand-induced signal can be detected within thirty minutes using high picomolar to low nanomolar concentrations of estrogen. As a result of the sensitivity (EC(50) for estradiol is approximately 0.7nM) and the very short assay time (2-4h) environmental water samples can typically be assayed directly without sterilization, extraction, and concentration. Thus, these assays represent rapid and sensitive approaches for determining the presence of contaminants in environmental samples. As proof of principle, we directly assayed wastewater influent and effluent taken from a wastewater treatment plant in the El Paso, TX area for the presence of estrogenic activity. The data obtained in the four-hour yeast bioassay directly correlated with GC-mass spectrometry analysis of these same water samples.