Highly stable peptide adducts from hard keratins as biomarkers to verify local sulfur mustard exposure of hair by high-resolution mass spectrometry.

In the recent past, the blister agent sulfur mustard (SM) deployed by the terroristic group Islamic State has caused a huge number of civilian and military casualties in armed conflicts in the Middle East. The vaporized or aerolized agent might be inhaled and have direct contact to skin and hair. Reaction products of SM with plasma proteins (adducts) represent well-established systemic targets for the bioanalytical verification of exposure. The SM-derived hydroxyethylthioethyl (HETE)-moiety is attached to nucleophilic amino acid side chains and allows unambiguous adduct detection. For shipping of common blood and plasma samples, extensive packaging rules are to be followed as these matrices are considered as potentially infectious material. In contrast, hair is considered as non-infectious thus making its handling and transportation much less complicated. Therefore, we addressed this matrix to develop a procedure for bioanalytical verification. Following optimized lysis of SM-treated human scalp hair and pepsin-catalyzed proteolysis of adducts of keratin type I and II, microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) was used to detect three alkylated keratin-derived biomarker peptides: AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF simultaneously. All bear the HETE-moiety bound to a glutamic acid residue. Protein adducts were stable for at least 14 weeks at ambient temperature and contact to air, and were not affected by washing the hair with shampoo. The biomarker peptides were also obtained from beard, armpit, abdominal, and pubic hair. This is the first report introducing stable local peptide adduct biomarkers from hair, that is easily accessible by a non-invasive sampling process.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 µM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

Post-translational modifications and stress adaptation: the paradigm of FKBP51.

Adaptation to stress is a fundamental requirement to cope with changing environmental conditions that pose a threat to the homeostasis of cells and organisms. Post-translational modifications (PTMs) of proteins represent a possibility to quickly produce proteins with new features demanding relatively little cellular resources. FK506 binding protein (FKBP) 51 is a pivotal stress protein that is involved in the regulation of several executers of PTMs. This mini-review discusses the role of FKBP51 in the function of proteins responsible for setting the phosphorylation, ubiquitination and lipidation of other proteins. Examples include the kinases Akt1, CDK5 and GSK3ß, the phosphatases calcineurin, PP2A and PHLPP, and the ubiquitin E3-ligase SKP2. The impact of FKBP51 on PTMs of signal transduction proteins significantly extends the functional versatility of this protein. As a stress-induced protein, FKBP51 uses re-setting of PTMs to relay the effect of stress on various signaling pathways.

Is Autophagy Involved in the Diverse Effects of Antidepressants?

Autophagy has received increased attention as a conserved process governing cellular energy and protein homeostasis that is thus relevant in a range of physiological and pathophysiological conditions. Recently, autophagy has also been linked to depression, mainly through its involvement in the action of antidepressants. Some antidepressant drugs and psychotropic medication have been reported to exert beneficial effects in other diseases, for example, in cancer and neurodegenerative diseases. This review collates the evidence for the hypothesis that autophagy contributes to the effects of antidepressants beyond depression treatment.

The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics.

Cytoskeletal dynamics are pivotal to memory, learning, and stress physiology, and thus psychiatric diseases. Downregulated in renal cell carcinoma 1 (DRR1) protein was characterized as the link between stress, actin dynamics, neuronal function, and cognition. To elucidate the underlying molecular mechanisms, we undertook a domain analysis of DRR1 and probed the effects on actin binding, polymerization, and bundling, as well as on actin-dependent cellular processes. METHODS: DRR1 domains were cloned and expressed as recombinant proteins to perform in vitro analysis of actin dynamics (binding, bundling, polymerization, and nucleation). Cellular actin-dependent processes were analyzed in transfected HeLa cells with fluorescence recovery after photobleaching (FRAP) and confocal microscopy. RESULTS: DRR1 features an actin binding site at each terminus, separated by a coiled coil domain. DRR1 enhances actin bundling, the cellular F-actin content, and serum response factor (SRF)-dependent transcription, while it diminishes actin filament elongation, cell spreading, and actin treadmilling. We also provide evidence for a nucleation effect of DRR1. Blocking of pointed end elongation by addition of profilin indicates DRR1 as a novel barbed end capping factor. CONCLUSIONS: DRR1 impacts actin dynamics in several ways with implications for cytoskeletal dynamics in stress physiology and pathophysiology.

Expression and glucocorticoid-dependent regulation of the stress-inducible protein DRR1 in the mouse adult brain.

Identifying molecular targets that are able to buffer the consequences of stress and therefore restore brain homeostasis is essential to develop treatments for stress-related disorders. Down-regulated in renal cell carcinoma 1 (DRR1) is a unique stress-induced protein in the brain and has been recently proposed to modulate stress resilience. Interestingly, DRR1 shows a prominent expression in the limbic system of the adult mouse. Here, we analyzed the neuroanatomical and cellular expression patterns of DRR1 in the adult mouse brain using in situ hybridization, immunofluorescence and Western blot. Abundant expression of DRR1 mRNA and protein was confirmed in the adult mouse brain with pronounced differences between distinct brain regions. The strongest DRR1 signal was detected in the neocortex, the CA3 region of the hippocampus, the lateral septum and the cerebellum. DRR1 was also present in circumventricular organs and its connecting regions. Additionally, DRR1 was present in non-neuronal tissues like the choroid plexus and ependyma. Within cells, DRR1 protein was distributed in a punctate pattern in several subcellular compartments including cytosol, nucleus as well as some pre- and postsynaptic specializations. Glucocorticoid receptor activation (dexamethasone 10 mg/kg s.c.) induced DRR1 expression throughout the brain, with particularly strong induction in white matter and fiber tracts and in membrane-rich structures. This specific expression pattern and stress modulation of DRR1 point to a role of DRR1 in regulating how cells sense and integrate signals from the environment and thus in restoring brain homeostasis after stressful challenges.

Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function.

The co-chaperone FKBP5 is a stress-responsive protein-regulating stress reactivity, and its genetic variants are associated with T2D related traits and other stress-related disorders. Here we show that FKBP51 plays a role in energy and glucose homeostasis. Fkbp5 knockout (51KO) mice are protected from high-fat diet-induced weight gain, show improved glucose tolerance and increased insulin signaling in skeletal muscle. Chronic treatment with a novel FKBP51 antagonist, SAFit2, recapitulates the effects of FKBP51 deletion on both body weight regulation and glucose tolerance. Using shorter SAFit2 treatment, we show that glucose tolerance improvement precedes the reduction in body weight. Mechanistically, we identify a novel association between FKBP51 and AS160, a substrate of AKT2 that is involved in glucose uptake. FKBP51 antagonism increases the phosphorylation of AS160, increases glucose transporter 4 expression at the plasma membrane, and ultimately enhances glucose uptake in skeletal myotubes. We propose FKBP51 as a mediator between stress and T2D development, and potential target for therapeutic approaches.

Prefrontal Cortex Corticotropin-Releasing Factor Receptor 1 Conveys Acute Stress-Induced Executive Dysfunction.

BACKGROUND: The medial prefrontal cortex (mPFC) subserves complex cognition and is impaired by stress. Corticotropin-releasing factor (CRF), through CRF receptor 1 (CRFR1), constitutes a key element of the stress response. However, its contribution to the effects of stress in the mPFC remains unclear. METHODS: Mice were exposed to acute social defeat stress and subsequently to either the temporal order memory (n = 11-12) or reversal learning (n = 9-11) behavioral test. Changes in mPFC Crhr1 messenger RNA levels were measured in acutely stressed mice (n = 12). Crhr1loxP/loxP mice received either intra-mPFC adeno-associated virus-Cre or empty microinjections (n = 17-20) and then were submitted to acute stress and later to the behavioral tests. Co-immunoprecipitation was used to detect activation of the protein kinase A (PKA) signaling pathway in the mPFC of acutely stressed mice (n = 8) or intra-mPFC CRF injected mice (n = 7). Finally, mice received intra-mPFC CRF (n = 11) and/or Rp-isomer cyclic adenosine 3',5' monophosphorothioate (Rp-cAMPS) (n = 12) microinjections and underwent behavioral testing. RESULTS: We report acute stress-induced effects on mPFC-mediated cognition, identify CRF-CRFR1-containing microcircuits within the mPFC, and demonstrate stress-induced changes in Crhr1 messenger RNA expression. Importantly, intra-mPFC CRFR1 deletion abolishes acute stress-induced executive dysfunction, whereas intra-mPFC CRF mimics acute stress-induced mPFC dysfunction. Acute stress and intra-mPFC CRF activate the PKA signaling pathway in the mPFC, leading to cyclic AMP response element binding protein phosphorylation in intra-mPFC CRFR1-expressing neurons. Finally, PKA blockade reverses the intra-mPFC CRF-induced executive dysfunction. CONCLUSIONS: Taken together, these results unravel a molecular mechanism linking acute stress to executive dysfunction via CRFR1. This will aid in the development of novel therapeutic targets for stress-induced cognitive dysfunction.

Azidobupramine, an Antidepressant-Derived Bifunctional Neurotransmitter Transporter Ligand Allowing Covalent Labeling and Attachment of Fluorophores.

The aim of this study was to design, synthesize and validate a multifunctional antidepressant probe that is modified at two distinct positions. The purpose of these modifications was to allow covalent linkage of the probe to interaction partners, and decoration of probe-target complexes with fluorescent reporter molecules. The strategy for the design of such a probe (i.e., azidobupramine) was guided by the need for the introduction of additional functional groups, conveying the required properties while keeping the additional moieties as small as possible. This should minimize the risk of changing antidepressant-like properties of the new probe azidobupramine. To control for this, we evaluated the binding parameters of azidobupramine to known target sites such as the transporters for serotonin (SERT), norepinephrine (NET), and dopamine (DAT). The binding affinities of azidobupramine to SERT, NET, and DAT were in the range of structurally related and clinically active antidepressants. Furthermore, we successfully visualized azidobupramine-SERT complexes not only in SERT-enriched protein material but also in living cells stably overexpressing SERT. To our knowledge, azidobupramine is the first structural analogue of a tricyclic antidepressant that can be covalently linked to target structures and further attached to reporter molecules while preserving antidepressant-like properties and avoiding radioactive isotopes.

The stress-inducible actin-interacting protein DRR1 shapes social behavior.

Understanding the molecular mechanisms by which stress is translated into changes in complex behavior may help to identify novel treatment strategies for stress-associated psychiatric disorders. The tumor suppressor gene down-regulated in renal cell carcinoma 1 (DRR1) was recently characterized as a new molecular link between stress, synaptic efficacy and behavioral performance, most likely through its ability to modulate actin dynamics. The lateral septum is one of the brain regions prominently involved in the stress response. This brain region features high DRR1 expression in adult mice, even under basal conditions. We therefore aimed to characterize and dissect the functional role of septal DRR1 in modulating complex behavior. DRR1 protein expression was shown to be expressed in both neurons and astrocytes of the lateral septum of adult mice. Septal DRR1 mRNA expression increased after acute defeat stress and glucocorticoid receptor activation. To mimic the stress-induced DRR1 increase in the lateral septum of mice, we performed adenovirus-mediated region-specific overexpression of DRR1 and characterized the behavior of these mice. Overexpression of DRR1 in the septal region increased sociability, but did not change cognitive, anxiety-like or anhedonic behavior. The observed changes in social behavior did not involve alterations of the expression of vasopressin or oxytocin receptors, the canonical social neuropeptidergic circuits of the lateral septum. In summary, our data suggest that the stress-induced increase of DRR1 expression in the lateral septum could be a protective mechanism to buffer or counterbalance negative consequences of stress exposure on social behavior.

N-desalkylquetiapine activates ERK1/2 to induce GDNF release in C6 glioma cells: a putative cellular mechanism for quetiapine as antidepressant.

Quetiapine is an atypical antipsychotic which has been suggested to possess also antidepressant efficacy in the treatment of bipolar and unipolar depression. Recently, a link between the activation of the ERK/MAPK signalling pathway and the release of GDNF has been proposed as a specific feature of antidepressants. To obtain a first insight into the putative molecular mechanism of action of quetiapine, we examined its impact and that of its major metabolite norquetiapine on the activation of the ERK/MAPK signalling pathway in C6 glioma cells. Additionally, we investigated the induction of GDNF release as a possible physiological consequence of this activation. We found that norquetiapine, similarly to the antidepressant reboxetine, activated both ERK1 and ERK2 (pERK) with consequent enhanced release of GDNF; this release was dependent on pERK, as demonstrated by its reversibility after pre-treatment with a pharmacological pERK inhibitor. In contrast, quetiapine induced activation of ERK2 only. It also caused release of GDNF, but this release was independent of ERK activation. To test whether the simultaneous activation of ERK1 with ERK2 was critical for the observed pERK-dependent GDNF release, we specifically inactivated ERK1 mRNA via RNA interference. Our data show that indeed ERK1 plays an essential role, as GDNF release was hampered after Erk1 downregulation comparably to a pharmacological pERK inhibitor. Thus, activation of only ERK2 appears not to be sufficient for promoting GDNF release. Our results reveal the release of GDNF as a consequence of ERK/MAPK signalling activation by norquetiapine, which may contribute to the putative antidepressant properties of quetiapine. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Tumor suppressor down-regulated in renal cell carcinoma 1 (DRR1) is a stress-induced actin bundling factor that modulates synaptic efficacy and cognition.

Stress has been identified as a major causal factor for many mental disorders. However, our knowledge about the chain of molecular and cellular events translating stress experience into altered behavior is still rather scant. Here, we have characterized a murine ortholog of the putative tumor suppressor gene DRR1 as a unique stress-induced protein in brain. It binds to actin, promotes bundling and stabilization of actin filaments, and impacts on actin-dependent neurite outgrowth. Endogenous DRR1 localizes to some, but not all, synapses, with preference for the presynaptic region. Hippocampal virus-mediated enhancement of DRR1 expression reduced spine density, diminished the probability of synaptic glutamate release, and altered cognitive performance. DRR1 emerges as a protein to link stress with actin dynamics, which in addition is able to act on synaptic function and cognition.

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 Alzheimer's 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.

Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors.

BACKGROUND: Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet. METHODOLOGY AND PRINCIPAL FINDINGS: We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action. CONCLUSION AND SIGNIFICANCE: The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity.

Impact of lipid raft integrity on 5-HT3 receptor function and its modulation by antidepressants.

Because of the biochemical colocalization of the 5-HT(3) receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT(3) receptor function and its modulation by antidepressants. Treatment with methyl-beta-cyclodextrine (MbetaCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MbetaCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT(3) receptor were retained. Although both the 5-HT(3) receptor and flotillin-1 were predominantly found in raft-like domains in western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT(3) receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT(3) receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT(3) receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel.

Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

DNA methylation dependent silencing of the human glutamate transporter EAAT2 gene in glial cells.

Glutamate is the major excitatory neurotransmitter in the CNS that is cleared from the extracellular space by a family of high-affinity glutamate transporters. The astroglial glutamate transporter EAAT2 is thought to carry out the uptake of the vast quantity of glutamate, and dysregulation of EAAT2 expression is involved in the pathogenesis of neurological disorders with marked excitotoxic components. Here, we present a novel epigenetic mechanism by which the human EAAT2 gene is kept in a silent state. Sequence inspection identified a classical CpG island at the EAAT2 promoter. Bisulfite analysis of the DNA methylation profile revealed that lack of EAAT2 expression in human glioma cell lines was associated with a densely methylated EAAT2 promoter. In contrast, EAAT2 positive normal human brain tissue used as reference displayed hypomethylation of the same promoter regions. In vitro methylation of EAAT2 promoter sequences indeed altered the binding properties of nuclear factors to the respective DNA sites as illustrated by electrophoretic mobility shift assay. Moreover, we observed a reduced activity of a methylated EAAT2 promoter construct as compared to the unmethylated control, both in a human glioma cell line and rodent primary astrocytes. Further supporting a role of DNA methylation for EAAT2 silencing, inhibition of DNA methyltransferases robustly enhanced EAAT2 mRNA transcription in several cell lines tested. In conclusion, the idea is put forward of an epigenetic mode of EAAT2 regulation based on the differential methylation of the gene promoter. (c) 2007 Wiley-Liss, Inc.

FK506-binding proteins 51 and 52 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor in mammalian cells.

We used a cellular system to elucidate the molecular determinants of the large immunophilin FK506-binding proteins (FKBP)51 and -52 for their action on the glucocorticoid receptor in mammalian cells. Increasing the levels of FKBP51 reduced the transcriptional activity of the receptor, as reported. Elevated levels of FKBP52 per se showed no effect but mitigated the inhibition of the receptor induced by FKBP51. We discovered that nuclear translocation of the glucocorticoid receptor was delayed by FKBP51. This correlates with the reduced interaction of FKBP51 with the motor protein dynein compared with FKBP52. From mutational analyses, we concluded that three features of the immunophilins are required for efficient receptor signaling in mammalian cells: hsp90 interaction, dynein association, and peptidylprolyl isomerase (PPIase) enzyme activity. The relevance of dynein for receptor function was substantiated by several experiments: 1) coexpression of dynamitin, which disrupts the transport complex and reduces receptor activity; 2) coexpression of the PPIase domain fragment of FKBP52, which is known to disrupt interaction of the receptor to dynein and reduce glucocorticoid receptor function, in contrast to the corresponding fragment of FKBP51; and 3) swapping of the PPIase domains FKBP51 and FKBP52, which reverses the respective activity. We concluded from our results that the mechanisms of the regulatory system FKBP51/FKBP52 discovered in yeast also operate in mammals to modulate hormone binding of the receptor. In addition, differential regulation of dynein association and nuclear translocation contributes to the effects of the two immunophilins on the glucocorticoid receptor in mammals.

Cofilin 1 is revealed as an inhibitor of glucocorticoid receptor by analysis of hormone-resistant cells.

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance.