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.

Transport and Golgi organisation protein 1 is a novel tumour progressive factor in oral squamous cell carcinoma.

Transport and Golgi organisation protein 1 (TANGO), also known as MIA3, belongs to the melanoma inhibitory activity (MIA) gene family. Although MIA acts as an oncogene, MIA2 and TANGO have a tumour-suppressive function in several malignancies; accordingly, the role and function of the MIA gene family in tumours remain controversial. Here the roles of TANGO were investigated in oral squamous cell carcinoma (OSCC). We analysed expression and function of TANGO in human OSCC cell lines. TANGO expression was also examined in 171 cases of primary OSCC by immunohistochemistry and statistically assessed the correlation between TANGO positivity and the clinicopathological parameters including vessel density. By TANGO knockdown in OSCC cells, the growth and invasion were repressed and apoptosis was induced. Activities of platelet-derived growth factor beta polypeptide (PDGFB) and Neuropilin2 were inhibited by TANGO knockdown. TANGO immunoreactivity was detected in 35.1% (60/171) cases of OSCC. TANGO expression was strongly associated with tumour progression, nodal metastasis, clinical stage and number of blood or lymph vessels in OSCC. Patients showing TANGO-expression fared significantly worse disease-free survival than cases without TANGO expression. These findings suggest that TANGO might promote angiogenesis and lymphangiogenesis by upregulation of PDGFB and Neuropilin2 in OSCC.

Semaphorins in angiogenesis and tumor progression.

The semaphorins were initially described as axon guidance factors, but have recently been implicated in a variety of physiological and developmental functions, including regulation of immune response, angiogenesis, and migration of neural crest cells. The semaphorin family contains more than 30 genes divided into seven subfamilies, all of which are characterized by the presence of a sema domain. The semaphorins transduce their signals by binding to one of the nine receptors belonging to the plexin family, or, in the case of the class 3 semaphorins, by binding to one of the two neuropilin receptors. Additional receptors, which form complexes with these primary semaphorin receptors, are also frequently involved in semaphorin signaling. Recent evidence suggests that some semaphorins can act as antiangiogenic and/or antitumorigenic agents whereas other semaphorins promote tumor progression and/or angiogenesis. Furthermore, loss of endogenous inhibitory semaphorin expression or function on one hand, and overexpression of protumorigenic semaphorins on the other hand, is associated with the progression of some tumor types.

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.

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy.

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ERalpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERalpha phosphorylation on Ser(118) in response to 17beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

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.

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.

Plant immunophilins: functional versatility beyond protein maturation.

Originally identified as the cellular targets of immunosuppressant drugs, the immunophilins encompass two ubiquitous protein families: the FK-506 binding proteins or FKBPs, and the cyclosporin-binding proteins or cyclophilins. Present in organisms ranging from bacteria to animals and plants, these proteins are characterized by their enzymatic activity; the peptidyl-prolyl cis-trans isomerization of polypeptides. Whilst this function is important for protein folding, it has formed the functional basis for more complex interactions between immunophilins and their target proteins. Beginning with a brief historical overview of the immunophilin family, and a representative illustration of the current state of knowledge that has accumulated for these proteins in diverse organisms, a detailed description is presented of the recent advances in the elucidation of the role of this ubiquitous protein family in plant biology. Though still in its infancy, investigation into the function of plant immunophilins has so far yielded interesting results--as a significant component of the chloroplast proteome, the abundance of immunophilins located in the thylakoid lumen suggests that these proteins may play important roles in this relatively uncharacterized subcellular compartment. Moreover, the importance of the complex multidomain immunophilins in functions pertaining to development is underscored by the strong phenotypes displayed by their corresponding mutants.

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.

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.

Molecular basis of anti-apoptotic effect of immunophilin ligands on hydrogen peroxide-induced apoptosis in human glioma cells.

To clarify the molecular basis of the cytoprotective properties of immunophilin ligands (IPLs), the anti-apoptotic effects of IPLs were determined in human glioma U251 cells. GPI1046 and V10367, non-immunosuppressive IPLs (NI-IPLs), as well as FK506, an immunosuppressive IPL (I-IPL), had cytoprotective effects against hydrogen peroxide (H20O)-induced apoptotic cell death in U251 cells. H2O2 increased both the ratio of bax/bcl-2 and the p53 mRNA expression. However, pre-treatment with FK506 and V10367 significantly prevented any increase in this ratio or p53 mRNA expression. GPI1046 also reduced the ratio of bax/bcl-2 to the normal level. In addition, H2O2 significantly increased activities of all three caspases, caspase-3, caspase-8, and caspase-9, in comparison with non-H2O2 controls. However, FK506 prevented the increase of these caspase activities. On the other hand, it is well-known that glutathione (GSH) and neurotrophic factor (NTF) is related to the induction of apoptosis in neuronal cells. In U251 cells, FK506, GPI1046 and V10367 had GSH-activating and NTF-activating effects. Thus, the immunosuppressive effect is not essential for the cytoprotective properties of IPLs, and IPLs have multiple beneficial properties such as the anti-apoptotic effect, GSH-activating effect, and NTF-activating effect, although the anti-apoptotic effect of NI-IPLs is independent of the regulation of apoptotic activators such as caspase-3.

Structural tolerance of bacterial autotransporters for folded passenger protein domains.

In this report we investigate the capacity of bacterial autotransporters (AT) to translocate folded protein domains across the outer membrane (OM). Polypeptides belonging to the AT family contain a C-terminal domain that supports the secretion of the N-domain (the passenger) across the OM of Gram-negative bacteria. Despite some controversial data, it has been widely accepted that N-passenger domains of AT must be unfolded and devoid of disulphide bonds for efficient translocation. To address whether or not AT are able to translocate folded protein domains across the OM, we employed several types of recombinant antibodies as heterologous N-passengers of the transporter C-domain of IgA protease (C-IgAP) of Neisseria gonorroheae. The N-domains used were single chain Fv fragments (scFv) and variable mono-domains derived from camel antibodies (V(HH)) selected on the basis of their distinct and defined folding properties (i.e. enhanced solubility, stability and presence or not of disulphide bonds). Expression of these hybrids in Escherichia coli shows that stable scFv and V(HH) domains are efficiently (>99%) translocated towards the bacterial surface regardless of the presence or not of disulphide bonds on their structure. Antigen-binding assays demonstrate that surface-exposed scFv and V(HH) domains are correctly folded and thus able to bind their cognate antigens. Expression of scFv- or V(HH)-C-IgAP hybrids in E. coli dsbA or fkpA mutant cells reveals that these periplasmic protein chaperones fold these N-domains before their translocation across the OM. Furthermore, large N-passengers composed of strings of V(HH) domains were secreted in a folded state by AT with no loss of efficacy (>99%) despite having multiple disulphide bonds. Thus AT can efficiently translocate toward the cell surface folded N-passengers composed of one, two or three immunoglobulin (Ig) domains, each with a folded diameter between approximately 2 nm and having disulphide bonds. This tolerance for folded protein domains of approximately 2 nm fits with the diameter of the central hydrophilic channel proposed for the ring-like oligomeric complex assembled by C-IgAP in the OM.

The FKBP12-rapamycin-associated protein (FRAP) is a CLIP-170 kinase.

CLIP-170/Restin belongs to a family of conserved microtubule (MT)-associated proteins, which are important for MT organization and functions. CLIP-170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP-170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12-rapamycin-associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP-170. CLIP-170 is phosphorylated in vivo at multiple sites, including rapamycin-sensitive and -insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin-sensitive sites. In addition, rapamycin inhibited the ability of CLIP-170 to bind to MTs. Our observations suggest that multiple CLIP-170 kinases are involved in positive and negative control of CLIP-170, and FRAP is a CLIP-170 kinase positively regulating the MT-binding behavior of CLIP-170.

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.

FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization.

Transforming growth factor-beta (TGF-beta) family polypeptides regulate cell growth and differentiation by binding to single pass serine/threonine kinases referred to as TGF-beta type I and II receptors. Although interaction screens have shown that the immunophilin FKBP12 interacts with TGF-beta type I receptors, the role of FKBP12 in TGF-beta receptor action is presently unclear. Using a chimeric TGF-beta receptor system, we have shown a specific enhancement of internalization when FKBP12 binding to the type I receptor was prevented with rapamycin. Moreover, although earlier studies demonstrated that type II receptor kinase activity was required for optimal internalization in mesenchymal cells, we found that rapamycin functioned downstream of the type II receptor kinase. Thus, rather than modulating TGF-beta signaling, our data suggest a novel role for FKBP12 as a negative regulator of TGF-beta receptor endocytosis.

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.

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.