A heat shock protein 90 inhibitor that modulates the immunophilins and regulates hormone receptors without inducing the heat shock response.

When a cell encounters external stressors, such as lack of nutrients, elevated temperatures, changes in pH or other stressful environments, a key set of evolutionarily conserved proteins, the heat shock proteins (hsps), become overexpressed. Hsps are classified into six major families with the hsp90 family being the best understood; an increase in cell stress leads to increased levels of hsp90, which leads to cellular protection. A hallmark of hsp90 inhibitors is that they induce a cell rescue mechanism, the heat shock response. We define the unique molecular profile of a compound (SM145) that regulates hormone receptor protein levels through hsp90 inhibition without inducing the heat shock response. Modulation of the binding event between heat shock protein 90 and the immunophilins/homologs using SM145, leads to a decrease in hormone receptor protein levels. Unlike N-terminal hsp90 inhibitors, this hsp90 inhibitor does not induce a heat shock response. This work is proof of principle that controlling hormone receptor expression can occur by inhibiting hsp90 without inducing pro-survival protein heat shock protein 70 (hsp70) or other proteins associated with the heat shock response. Innovatively, we show that blocking the heat shock response, in addition to hsp90, is key to regulating hsp90-associated pathways.

The emerging role of FK506-binding proteins as cancer biomarkers: a focus on FKBPL.

FKBPs (FK506-binding proteins) have long been recognized as key regulators of the response to immunosuppressant drugs and as co-chaperones of steroid receptor complexes. More recently, evidence has emerged suggesting that this diverse protein family may also represent cancer biomarkers owing to their roles in cancer progression and response to treatment. FKBPL (FKBP-like) is a novel FKBP with roles in GR (glucocorticoid receptor), AR (androgen receptor) and ER (oestrogen receptor) signalling. FKBPL binds Hsp90 (heat-shock protein 90) and modulates translocation, transcriptional activation and phosphorylation of these steroid receptors. It has been proposed as a novel prognostic and predictive biomarker, where high levels predict for increased recurrence-free survival in breast cancer patients and enhanced sensitivity to endocrine therapy. Since this protein family has roles in a plethora of signalling pathways, its members represent novel prognostic markers and therapeutic targets for cancer diagnosis and treatment.

Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis.

Here, we describe the isolation of adenine nucleotide translocase-1 (ANT-1) in a screen for dominant, apoptosis-inducing genes. ANT-1 is a component of the mitochondrial permeability transition complex, a protein aggregate connecting the inner with the outer mitochondrial membrane that has recently been implicated in apoptosis. ANT-1 expression led to all features of apoptosis, such as phenotypic alterations, collapse of the mitochondrial membrane potential, cytochrome c release, caspase activation, and DNA degradation. Both point mutations that impair ANT-1 in its known activity to transport ADP and ATP as well as the NH(2)-terminal half of the protein could still induce apoptosis. Interestingly, ANT-2, a highly homologous protein could not lead to cell death, demonstrating the specificity of the signal for apoptosis induction. In contrast to Bax, a proapoptotic Bcl-2 gene, ANT-1 was unable to elicit a form of cell death in yeast. This and the observed repression of apoptosis by the ANT-1-interacting protein cyclophilin D suggest that the suicidal effect of ANT-1 is mediated by specific protein-protein interactions within the permeability transition pore.

Role of immunophilins in recovery of erectile function after cavernous nerve injury.

INTRODUCTION: Immunophilin ligands provide potentially new alternatives for the treatment of erectile dysfunction (ED), which occurs after injury of the cavernous nerves (CNs). AIM: To review and update current knowledge of the neurotrophic effects and likely mechanism of action of immunophilin proteins with emphasis on the FK506-binding protein (FKBP) subfamily and the role of immunophilin ligands for the treatment of CN injury-induced ED. METHODS: Review of available reports of studies investigating the effects and neurotrophic mechanisms of immunophilin ligands involved in erectile function recovery in rodent models of CN injury. MAIN OUTCOME MEASURES: Erection parameters and molecular correlations associated with CN injury and functional recovery. RESULTS: Treatment with prototype immunosuppressive immunophilin ligands FK506 (FK) and rapamycin (Rapa) improve erectile function in animal models of CN injury. Similarly, non-immunosuppressive analogs such as GPI-1046 and FK1706 are effective in recovery of erections after CN injury. Neuronal nitric oxide may influence the erection recovery effects of immunophilin ligands after CN injury. FKBPs 38 and 65 expression changes in the penis and its innervation coincide with the neurotrophic effects of immunophilin ligands. Antioxidative actions of immunophilin ligands contribute to their neurotrophic effects. Immunophilins are localized to nerves coursing in human prostate and penile tissue. CONCLUSIONS: The findings support the hypothesis that immunophilin ligands, working through specific receptor mechanisms that are specific to injured CN, are potentially useful to sustain erectile function in men following radical prostatectomy.

A novel FK506-like binding protein interacts with the glucocorticoid receptor and regulates steroid receptor signaling.

FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.

Spliceosomal immunophilins.

The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known proteinaceous splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis-trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data indicates that some members of this protein family are activators of spliceosomal proteins by way of folding and transport.

VEGF at the neurovascular interface: therapeutic implications for motor neuron disease.

VEGF was discovered almost 25 years ago, and its angiogenic activity has been extensively studied ever since. Accumulating evidence indicates, however, that VEGF also has direct effects on neuronal cells. VEGF exerts neuroprotective effects on various cultured neurons of the central nervous system. In vivo, VEGF controls the correct migration of facial branchiomotor neurons in the developing hindbrain and stimulates the proliferation of neural stem cells in enriched environments and after cerebral ischemia. Transgenic mice expressing reduced levels of VEGF develop late-onset motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis (ALS), whereas reduced levels of VEGF have been implicated in a polyglutamine-induced model of motor neuron degeneration. Recent data further reveal that intracerebroventricular delivery of recombinant VEGF protein delays disease onset and prolongs survival of ALS rats, whereas intramuscular administration of a VEGF-expressing lentiviral vector increases the life expectancy of ALS mice by as much as 30%. Deciphering the precise role of VEGF at the neurovascular interface promises to uncover new insights into the development and pathology of the nervous system, helpful to design novel strategies to treat (motor) neurodegenerative disorders.

Enzymes that catalyse the restructuring of proteins.

The large enzyme families of protein disulfide isomerases and peptidyl prolyl cis/trans isomerases have been shown to assist polypeptide restructuring. Various folding states of polypeptides may serve as substrates of the catalysed reaction. Our understanding of the cellular function of these enzymes is increasing as a result of the availability of more specific inhibitors, the discovery of natural substrates and the use of genetically modified organisms. Further highlights of these studies include insights into the three-dimensional structures of enzyme-ligand complexes, as well as into the mechanism of slow folding phases on the atomic level.

[Molecular mechanism of ischemic brain injuries and perspectives of drug therapies for neuroprotection].

Ischemic brain injury is a critical condition in the management of patients during anesthesia and intensive care. It is not rare that pathological conditions such as cerebral ischemia, head trauma and low oxygen result in marked impairment of cerebral function, even if the patient's life is saved. We sometimes encounter sudden changes in a patient's condition not only during anesthesia, but also in intensive care unit with transient low-oxygen and ischemic conditions accompanying serious shock. We have been studying the mechanisms to counteract pathological conditions leading to neuronal cell death that have been exposed to such emergency conditions, and to discover therapeutic methods to minimize the brain damage after insult. With advances in the understanding of the mechanism of neuronal cell death, technology in intensive care for salvaging neuronal cell that are at the brink of death and for recovery of brain function has progressed. However, a breakthrough has not been achieved in the development of effective therapy. Protection of the brain from terminal impairment and preservation of function will be an important issue. To achieve this goal, it is critical to clarify the susceptible mechanisms causing ischemic brain damage. This report discusses the importance of the calcineurin/immunophilin signal transduction mechanism as a new mechanism that is involved in the induction of ischemic brain damage and refers the status-quo of cerebral protection by drug therapy.

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action.

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.

Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.

Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy.

Chloroplast immunophilins.

Immunophilins occur in almost all living organisms. They are ubiquitously expressed proteins including cyclophilins, FK506/rapamycin-binding proteins, and parvulins. Their functional significance in vascular plants is mostly related to plant developmental processes, signalling, and regulation of photosynthesis. Enzymatically active immunophilins catalyse isomerization of proline imidic peptide bonds and assist in rapid folding of nascent proline-containing polypeptides. They also participate in protein trafficking and assembly of supramolecular protein complexes. Complex immunophilins possess various additional functional domains associated with a multitude of molecular interactions. A considerable number of immunophilins act as auxiliary and/or regulatory proteins in highly specialized cellular compartments, such as lumen of thylakoids. In this review, we present a comprehensive overview of so far identified chloroplast immunophilins that assist in specific assembly/repair processes necessary for the maintenance of efficient photosynthetic energy conversion.

The immunophilin ligand FK506, but not the P38 kinase inhibitor SB203580, improves function of adult rat muscle reinnervated from transplants of embryonic neurons.

Injury to the adult CNS often involves death of motoneurons, resulting in the paralysis and progressive atrophy of muscle. There is no effective therapy to replace motoneurons in the CNS. Our strategy to replace neurons and to rescue denervated muscles is to transplant dissociated embryonic day 14-15 (E14-15) ventral spinal cord cells into the distal stump of a peripheral nerve near the denervated muscles. Here, we test whether long-term delivery of two pharmacological inhibitors to denervated muscle, FK506 or SB203580, enhances reinnervation of muscle from embryonic cells transplanted in the tibial nerve of adult Fischer rats. FK506, SB203580 (2.5 mg/kg) or saline was delivered under the fascia of the medial gastrocnemius muscle for 4 weeks, beginning when muscles were denervated by section of the sciatic nerve. After 1 week of nerve degeneration, one million E14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of each rat in the three treatment groups. Ten weeks later, all cell transplants had neuron-specific nuclear protein (NeuN) positive neurons. Neuron survival and axon regeneration were similar across treatments. An average (+/-S.E.) of 210+/-66, 100+/-36 and 176+/-58 myelinated axons grew distally from the cell transplants of rats with muscles treated with FK506, SB203580 or saline, respectively. Regenerating axons in muscles of all three treatments groups were detected with antibodies against phosphorylated neurofilaments and synaptophysin, and motor end plates were labeled with alpha-bungarotoxin. Muscles of rats that received transplants of media only had no axon growth, indicating that the muscles were denervated. The mean muscle fiber areas of rats that received cell transplants and had long-term delivery of FK506, SB203580 or saline to muscles were significantly larger than those of denervated muscle fibers. Thus, cell transplantation reduced muscle atrophy. Transplantation of embryonic cells also resulted in functional muscle reinnervation. Electromyographic activity and force were evoked from >90% of the muscles of rats with cell transplants, but not from denervated muscles. FK506-treated muscles were significantly more fatigue resistant than naive control muscles. FK506-treated muscles also had significantly stronger motor units than those in SB203580 or saline-treated muscles. These data suggest that a pathway regulated by FK506 improves the function of muscles reinnervated by embryonic neurons placed in peripheral nerve.

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase.

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.

Oxidative stress in the molecular mechanism of pathogenesis at different diseased states of organism in clinics and experiment.

According to modern images and results of our observations the oxidative stress (OS) is a non-specific though certain component of pathogenesis at numerous diseased states of organism having in the basis the thoroughness of pathogenic disturbances of phospholipids (PL) metabolism and processes of their free radical oxidation (FRO), which takes place in the membrane formations of as the whole cell, as well as the mitochondrial and microsomal fractions (MCF and MSF) of the white rat brain, liver mitochondria, lung shadows, at the same time erythrocyte and lymphocyte shadows at brain acute edema, ischemia, reperfusion and desympathization, infarction of myocardium, tuberculosis of lungs, diabetes, Familial Mediterranean Fever (FMF), intoxications under halothane anaesthesia (HA) and with micotoxin zearalenon. The regularities observed promote to understand from the point of view of modern approaches the molecular mechanisms of initiation, development and generalization of factors for OS formation under pathologic conditions. It is more obvious at zearalenon intoxication with intensification of lipids FRO processes and failures in PL-PL ratio phenomena. The lymphocytes membranes of the white rats spleen subjected OS induced by zearalenon intoxication permit us conclude that the general immune status of the organism decreases. It is generally peculiar to the states under conditions of generalized intoxication. The observed increase of phospholipase A(2) activity induces the release of high concentrations of lysophosphatidylcholines (LPC) and non-etherified fatty acids (NEFA) of polyenic range with prevail of arachidonic acid as a pathogenic factor, namely, at modelling brain acute edema by tetraethylolovo to white rats. Formation of the above mentioned disturbances to some extent depends on hydrophobic properties of toxins, particularly, zearalenon. The latter gives certain tropism to dopamine-beta-monooxygenase (DBM), and ability to stimulate functional activity of the enzyme. Striking haemolytic properties of phospholipase A(2) induced by existence of LPC and NEFA high concentrations, and products of their peroxidation, promote elimination of separate protein fractions of erythrocyte membranes (EM) responsible for OS formation and decrease of erythrocytes resistance to peroxide hemolysis. Increase of DBM activity under the effect of relatively moderate doses of zearalenon (1-15 microg/ml) is accompanied with extra intensification of catecholamine synthesizing function of the organism with lethal result. Data of publications represented testify exceptional efficiency of sodium thiosulfate (STS) as a powerful synergist for endogenous factors of antioxidant effect, particularly alpha-tocopherol (alpha-T), which is the main component for the system of cell antiradical defence. Detoxicating effect of STS can be demonstrated indeed on the example of zearalenon intoxication during the first two hours with the reduction of metabolism disturbances of PL and products of its peroxidation. Comparative evaluation of molecular mechanisms of STS normalizing effect as a supplier for hydrogen and sulphur ions, as well as an effective synergist for alpha-T on the level of various formations of the live cell in compare with the effects of alpha-T and ubiquinone, allowed to make a special accent on the role of STS in interaction with energy-dependent enzymatic systems of cell antiradical defence, as well as accumulation and transformation of energy on the level of mitochondrial membranes. The results obtained by us confirm a number of clinical experimental observations, which demonstrate treatment and prophylactic role of STS at different pathologic states of the organism. STS protectory role at toxic injuries of the organism is higher at its preliminary introduction to the organism before modelling of the studied diseased states, especially at zearalenon and halothane (H) intoxication (in the last case before HA). These data serve a sound affirmation for protectory function of STS, detailed revelation of molecular properties of pathogenesis of the studied intoxication to which a part of our clinical and experimental studies at present is devoted.

Induction of differentiation of human myeloid leukemia cells by immunosuppressant macrolides (rapamycin and FK506) and calcium/calmodulin-dependent kinase inhibitors.

OBJECTIVE: Potent immunosuppressants, such as rapamycin, FK506, and ascomycin, are known to regulate the phosphorylation of proteins. The purpose of this study was to investigate the effects of these immunosuppressants on differentiation of several human myeloid leukemic cell lines. MATERIALS AND METHODS: Human myeloid leukemic cell lines were cultured with each immunosuppressant, and several differentiation markers were assayed. RESULTS: Rapamycin effectively induced granulocytic differentiation of human myeloid leukemic HL-60 and ML-1 cells. In addition to morphologic differentiation, it also induced nitroblue tetrazolium reduction, lysozyme activity, and expression of CD11b in HL-60 cells. The commitment to differentiation was observed after treatment with rapamycin for 1 day, indicating that the effect of rapamycin was irreversible. FK506 and ascomycin induced differentiation of HL-60 cells, but at higher concentrations than rapamycin. A calcium/calmodulin-dependent kinase (CaMK) was copurified with FKBP52 immunophilin, a binding protein of immunosuppressants. We also found that the CaMK inhibitors KN62 and KN93 induced differentiation of HL-60 cells. Rapamycin and CaMK inhibitors induced differentiation of human myeloid leukemia ML-1 and K562, but not of other cell lines such as NB4, U937, or HEL. CONCLUSION: Immunosuppressants and CaMK inhibitors induced differentiation of HL-60, ML-1, and K562 cells.

FRAP DNA-dependent protein kinase mediates a late signal transduced from ultraviolet-induced DNA damage.

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation.

Archaeal peptidyl prolyl cis-trans isomerases (PPIases).

PPIases are ubiquitous in living organisms. While 3 families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases. Among 2 cyclophilins found in Archaea, only Halobacterium cyclophilin (HcCyP19) has been characterized. It is a cyclosporin A (CsA) sensitive CyP with a MW of 19.4kDa. The PPIase activity and CsA sensitivity of this CyP is higher at higher salt concentration in the medium. No parvulin or its homolog has been found in Archaea. Two types of FKBPs, 26-30kDa long type and 17-18 kDa short type FKBP, have been found in Archaea. While the N-terminal regions of these 2 type FKBPs are similar to each other, the long type archaeal FKBP has an additional ca. 100 amino-acid sequence at its C-terminal region. In comparison with human HsFKBP12, the N-terminal region of the archaeal FKBP has 2 insertion sequences in the regions corresponding to Bulge and Flap of HsFKBP12. A short type archaeal FKBP from Methanococcus thermolithotrophicus has been shown to have not only a PPIase activity but also a chperone like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates. Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the Flap seemed to be important.

Immunophilin chaperones in steroid receptor signalling.

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Structures of immunophilins and their ligand complexes.

This review includes an analysis of available X-ray and NMR structures of both members of the immunophilin family; cyclophilins and the FK-506 binding proteins (FKBPs). Available structures are compared and contrasted to highlight different structural features seen both within and between species. Each immunophilin family has been structurally characterised with a variety of small molecule ligands, principally immunosuppressive drugs and their analogues and an overview of these complexes is also presented. Currently the Protein Data Base contains over 60 entries for cyclophilins and over 40 entries for FKBPs. A number of FKBP related structures are also available including structures of MIP (Macrophage Infectivity Potentiator protein) from Legionella pneumophila and Trypanosoma cruzi and Trigger Factor from Mycoplasma genitalium. For all structures discussed in the review a summary of the available biological data is also presented.