Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

BACKGROUND

In addition to trauma exposure, other factors contribute to risk for development of posttraumatic stress disorder (PTSD) in adulthood. Both genetic and environmental factors are contributory, with child abuse providing significant risk liability.

OBJECTIVE

To increase understanding of genetic and environmental risk factors as well as their interaction in the development of PTSD by gene x environment interactions of child abuse, level of non-child abuse trauma exposure, and genetic polymorphisms at the stress-related gene FKBP5.

METHODS

A cross-sectional study examining genetic and psychological risk factors in 900 nonpsychiatric clinic patients (762 included for all genotype studies) with significant levels of childhood abuse as well as non-child abuse trauma using a verbally presented survey combined with single-nucleotide polymorphism (SNP) genotyping. Participants were primarily urban, low-income, black (>95%) men and women seeking care in the general medical care and obstetrics-gynecology clinics of an urban public hospital in Atlanta, Georgia, between 2005 and 2007.

METHODS

Severity of adult PTSD symptomatology, measured with the modified PTSD Symptom Scale, non-child abuse (primarily adult) trauma exposure and child abuse measured using the traumatic events inventory and 8 SNPs spanning the FKBP5 locus.

RESULTS

Level of child abuse and non-child abuse trauma each separately predicted level of adult PTSD symptomatology (mean [SD], PTSD Symptom Scale for no child abuse, 8.03 [10.48] vs>> or =2 types of abuse, 20.93 [14.32]; and for no non-child abuse trauma, 3.58 [6.27] vs>> or =4 types, 16.74 [12.90]; P < .001). Although FKBP5 SNPs did not directly predict PTSD symptom outcome or interact with level of non-child abuse trauma to predict PTSD symptom severity, 4 SNPs in the FKBP5 locus significantly interacted (rs9296158, rs3800373, rs1360780, and rs9470080; minimum P = .0004) with the severity of child abuse to predict level of adult PTSD symptoms after correcting for multiple testing. This gene x environment interaction remained significant when controlling for depression severity scores, age, sex, levels of non-child abuse trauma exposure, and genetic ancestry. This genetic interaction was also paralleled by FKBP5 genotype-dependent and PTSD-dependent effects on glucocorticoid receptor sensitivity, measured by the dexamethasone suppression test.

CONCLUSIONS

Four SNPs of the FKBP5 gene interacted with severity of child abuse as a predictor of adult PTSD symptoms. There were no main effects of the SNPs on PTSD symptoms and no significant genetic interactions with level of non-child abuse trauma as predictor of adult PTSD symptoms, suggesting a potential gene-childhood environment interaction for adult PTSD.

Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders.

As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

The stress hormone-regulating hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the causality as well as the treatment of depression. To investigate a possible association between genes regulating the HPA axis and response to antidepressants and susceptibility for depression, we genotyped single-nucleotide polymorphisms in eight of these genes in depressed individuals and matched controls. We found significant associations of response to antidepressants and the recurrence of depressive episodes with single-nucleotide polymorphisms in FKBP5, a glucocorticoid receptor-regulating cochaperone of hsp-90, in two independent samples. These single-nucleotide polymorphisms were also associated with increased intracellular FKBP5 protein expression, which triggers adaptive changes in glucocorticoid receptor and, thereby, HPA-axis regulation. Individuals carrying the associated genotypes had less HPA-axis hyperactivity during the depressive episode. We propose that the FKBP5 variant-dependent alterations in HPA-axis regulation could be related to the faster response to antidepressant drug treatment and the increased recurrence of depressive episodes observed in this subgroup of depressed individuals. These findings support a central role of genes regulating the HPA axis in the causality of depression and the mechanism of action of antidepressant drugs.

FK506 binding protein 51 or FKBP5 is a co-chaperone of hsp90 which regulates glucocorticoid receptor (GR) sensitivity. When it is bound to the receptor complex, cortisol binds with lower affinity and nuclear translocation of the receptor is less efficient. FKBP5 mRNA and protein expression are induced by GR activation via intronic hormone response elements and this provides an ultra-short feedback loop for GR-sensitivity. Polymorphisms in the gene encoding this co-chaperone have been shown to associate with differential upregulation of FKBP5 following GR activation and differences in GR sensitivity and stress hormone system regulation. Alleles associated with enhanced expression of FKBP5 following GR activation, lead to an increased GR resistance and decreased efficiency of the negative feedback of the stress hormone axis in healthy controls. This results in a prolongation of stress hormone system activation following exposure to stress. This dysregulated stress response might be a risk factor for stress-related psychiatric disorders. In fact, the same alleles are over-represented in individuals with major depression, bipolar disorder and post-traumatic stress disorder. In addition, they are also associated with faster response to antidepressant treatment. FKBP5 might thus be an interesting therapeutic target for the prevention and treatment of stress-related psychiatric disorders.

We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

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.

We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR-Hsp90-FKBP52-dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNA-binding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport.

To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.

FKBP5 regulates the cortisol-binding affinity and nuclear translocation of the glucocorticoid receptor. Polymorphisms at the FKBP5 locus have been associated with increased recurrence risk of depressive episodes and rapid response to antidepressant treatment. A recent study showed that FKBP5 genotypes moderated the risk of post-traumatic stress disorder (PTSD) symptoms associated with childhood maltreatment. One thousand one hundred forty-three European Americans (EAs) and 1284 African Americans (AAs) recruited for studies of the genetics of substance dependence were also screened for lifetime PTSD. Four single-nucleotide polymorphisms (SNPs) in FKBP5, rs3800373, rs9296158, rs1360780, and rs9470080, were genotyped on the complete sample. Logistic regression analyses were performed to explore the interactive effect of FKBP5 polymorphisms and childhood adversity on the risk for PTSD. After correction for multiple testing, childhood adversity significantly increased the risk for PTSD. FKBP5 genotypes were not associated with the development of the disorder. In AAs, one of the SNPs, rs9470080, moderated the risk of PTSD that was associated with childhood abuse. Without childhood adverse experiences, participants with the TT genotype of this SNP had the lowest risk for PTSD, whereas they had the highest risk for PTSD after childhood adversity exposure. In addition, in EAs, alcohol dependence was observed to interact with childhood adverse experiences, and also FKBP5 polymorphisms, to increase the risk for PTSD. This study provides further evidence of a gene x environment effect of FKBP5 and childhood abuse on the risk for PTSD in AAs. Further study is required in other populations.

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Mood and anxiety disorders are considered stress-related diseases characterized by an impaired function of mineralocorticoid and glucocorticoid receptors (MR and GR, respectively), the major regulatory elements of the hypothalamus-pituitary-adrenocortical (HPA) axis. A number of so-called chaperone proteins moderate the function of these receptors. Genetic variations in one of these chaperones, FKBP5, were associated with antidepressant treatment response in depression and with a major risk-factor for the development of posttraumatic stress disorder. To further investigate the effect of FKPB5 polymorphisms on corticosteroid receptor-mediated HPA axis regulation we conducted the Trier Social Stress test, a standardized procedure to evaluate psychosocial stress response, in 64 healthy volunteers. We genotyped rs4713916, rs1360780 and rs3800737, the three single nucleotide polymorphisms (SNPs) in the FKBP5 region which had shown the strongest effect in previous studies. In addition, we evaluated the effects of the GR polymorphisms Bcl1 and N363S as well as the MR polymorphism I180V. Subjects homozygous for any of the FKBP5 variants displayed an incomplete normalization of the stress-elicited cortisol secretion. This was also observed following a second test additionally accompanied by an increased self-reported anxiety. Regarding GR and MR, only carriers of the Bcl1 variant displayed an altered cortisol response in the prognosticated direction. While Bcl1 was predominantly associated with anticipatory cortisol, homozygous carriers of the FKBP5 minor allele showed insufficient cortisol recovery and increased self-reported anxiety after psychosocial stress. This reaction pattern suggests that subjects carrying these variants are at risk of displaying chronically elevated cortisol levels after repeated stress constituting a risk factor for stress-related diseases.

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.

Childhood trauma is associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation and is a known risk factor for suicidal behavior. In this study we sought to determine whether the impact of childhood trauma on suicide risk might be modified by FKBP5, an HPA-axis regulating gene. Sixteen FKBP5 haplotype-tagging single nucleotide polymorphisms (SNPs) were genotyped in a sample of African Americans: 398 treatment-seeking patients with substance dependence (90% men; 120 suicide attempters) and 432 nonsubstance-dependent individuals (40% men; 21 suicide attempters). In all, 474 participants (112 suicide attempters) also completed the Childhood Trauma Questionnaire (CTQ). Primary haplotype analyses were conducted with the four SNPs implicated in earlier studies: rs3800373, rs9296158, rs1360780, and rs9470080. We found that childhood trauma was associated with suicide attempt (P<0.0001). Although there was no main effect of the two major yin yang haplotypes in the four SNP haplotype blocks, there was a haplotype influence on suicide risk (p=0.006) only in individuals exposed to high levels of childhood trauma. In this group, 51% with two copies of the risk haplotype, 36% with one copy, and 20% with no copies had attempted suicide. The total logistic regression model accounted for 13% of the variance in attempted suicide. Analyses of the 16 SNPs showed significant main effects on suicide attempt of rs3777747, rs4713902, and rs9470080 and interactive effects of rs3800373, rs9296158, and rs1360780 with CTQ score on suicide attempt. These data suggest that childhood trauma and variants of the FKBP5 gene may interact to increase the risk for attempting suicide.

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

Exposure to stressful events during development has consistently been shown to produce long-lasting alterations in the hypothalamic-pituitary-adrenal (HPA) axis, which may increase vulnerability to disease, including posttraumatic stress disorder and other mood and anxiety disorders. Recently reported genetic association studies indicate that these effects may be mediated, in part, by genexenvironment interactions involving polymorphisms within two key genes, CRHR1 and FKBP5. Data suggest that these genes regulate HPA axis function in conjunction with exposure to child maltreatment or abuse. In addition, a large and growing body of preclinical research suggests that increased activity of the amygdala-HPA axis induced by experimental manipulation of the amygdala mimics several of the physiological and behavioral symptoms of stress-related psychiatric illness in humans. Notably, interactions between the developing amygdala and HPA axis underlie critical periods for emotional learning, which are modulated by developmental support and maternal care. These translational findings lead to an integrated hypothesis: high levels of early life trauma lead to disease through the developmental interaction of genetic variants with neural circuits that regulate emotion, together mediating risk and resilience in adults.

BACKGROUND

In a recent study of several antidepressant drugs in hospitalized, non-Hispanic White patients, Binder et al. reported association of markers located within the FKBP5 gene with treatment response after 2 and 5 weeks. Individuals homozygous for the TT-genotype at one of the markers (rs1360780) reported more depressive episodes and responded better to antidepressant treatment. There was no association between markers in FKBP5 and disease. The present study aimed at studying the associated FKBP5 markers in the ethnically diverse Sequenced Treatment Alternatives to Relieve Depression (STAR*D) sample of non-hospitalized patients treated with citalopram.

METHODS

We used clinical data and DNA samples from 1809 outpatients with non-psychotic major depressive disorder (DSM-IV criteria), who received up to 14 weeks of citalopram. A subset of 1523 patients of White non-Hispanic or Black race was matched with 739 control subjects for a case-control analysis. The markers rs1360780 and rs4713916 were genotyped on the Illumina platform. TaqMan-assay was used for marker rs3800373.

RESULTS

In the case-control analysis, marker rs1360780 was significantly associated with disease status in the White non-Hispanic sample after correction for multiple testing. A significant association was also found between rs4713916 and remission. Markers rs1360780 and rs4713916 were in strong linkage disequilibrium in the White non-Hispanic but not in the Black population. There was no significant difference in the number of previous episodes of depression between genotypes at any of the three markers.

CONCLUSIONS

These results indicate that FKBP5 is an important target for further studies of depression and treatment response.

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P(4)) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P(4) binding to PR, attenuated PR transcriptional activity and down-regulation of several P(4)-regulated genes in uteri of Fkbp52(-/-) mice, establishes this cochaperone as a critical regulator of uterine P(4) function. Interestingly, ovulation, another P(4)-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.

The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90alpha (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the approximately 25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains.

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 Alzheimer's 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.

Expression of FKBP51, a large molecular weight immunophilin, is strongly enhanced by glucocorticoids, progestins, and androgens. However, the activity of a 3.4-kb fragment of the FKBP51 gene (FKBP5) promoter was only weakly increased by progestin and we show here that it is unresponsive to glucocorticoids and androgens. The entire FKBP5 was scanned for consensus hormone response elements (HREs) using MatInspector. We found that 2 regions of intron E, which are conserved in rat and mouse FKBP5, contain HRE-like sequences with high match scores. Deoxyribonucleic acid fragments (approximately 1 kb in length) containing these regions were amplified and tested in reporter gene assays for steroid responsiveness. One region of intron E of FKBP5 (pIE2) conferred both glucocorticoid and progestin responsiveness to 2 heterologous reporter genes, whereas the other, less-conserved region of intron E (pIE1) was responsive only to progestins. The inclusion of pIE1 upstream of pIE2 (pIE1IE2) enhanced progestin but not glucocorticoid responsiveness. None of the constructs containing intronic sequences was responsive to androgens. Mutation of the putative HREs within pIE1 and pIE2 eliminated hormone responsiveness. Electrophoretic mobility shift assays demonstrated that progesterone receptors (PR) bound to the HRE in pIE1, whereas both PR and glucocorticoid receptors interacted with the HRE in pIE2. These data suggest that distal intronic elements significantly contribute to transcriptional regulation of FKBP5 by glucocorticoids and progestins.