kDa Ligand Research Articles

FGIN-1-27 Mitigates Radiation-induced Mitochondrial Hyperfunction and Cellular Hyperactivation in Cultured Astrocytes

Radiation-induced brain injury (RBI) poses a significant challenge in the context of radiotherapy for intracranial tumors, necessitating a comprehensive understanding of the cellular and molecular mechanisms involved. While prior investigations have underscored the role of astrocyte activation and excessive vascular endothelial growth factor production in microvascular damage associated with RBI, there remains a scarcity of studies examining the impact of radiation on astrocytes, particularly regarding organelles such as mitochondria. Thus, our study aimed to elucidate alterations in astrocyte and mitochondrial functionality following radiation exposure, with a specific focus on evaluating the potential ameliorative effects of translocator protein 18 kDa(TSPO) ligands. In this study, cultured astrocytes were subjected to X-ray irradiation, and their cellular states and mitochondrial functions were examined and compared to control cells. Our findings revealed that radiation-induced astrocytic hyperactivation, transforming them into the neurotoxic A1-type, concomitant with reduced cell proliferation. Additionally, radiation triggered mitochondrial hyperfunction, heightened the mitochondrial membrane potential, and increased oxidative metabolite production. However, following treatment with FGIN-1-27, a TSPO ligand, we observed a restoration of mitochondrial function and a reduction in oxidative metabolite production. Moreover, this intervention mitigated astrocyte hyperactivity, decreased the number of A1-type astrocytes, and restored cell proliferative capacity. In conclusion, our study has unveiled additional manifestations of radiation-induced astrocyte dysfunction and validated that TSPO ligands may serve as a promising therapeutic strategy to mitigate this dysfunction. It has potential clinical implications for the treatment of RBI.

Differential effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on startle response to predictable threat in a NPU-threat task after acute and short-term treatment

RationaleBenzodiazepines have been extensively investigated in experimental settings especially after single administration, which mostly revealed effects on unpredictable threat (U-threat) rather than predictable threat (P-threat). Given the need for pharmacological alternatives with a preferable side-effect profile and to better represent clinical conditions, research should cover also other anxiolytics and longer application times.ObjectivesThe present study compared the acute and short-term effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on P-threat and U-threat while controlling for sedation.MethodsSixty healthy male volunteers, aged between 18 and 55 years, were randomly assigned to receive a daily dose of either 150 mg etifoxine, 1.5 mg alprazolam, or placebo for 5 days. On days 1 and 5 of intake, they performed a NPU-threat task including neutral (N), predictable (P), and unpredictable (U) conditions, while startle responsivity and self-reports were studied. Sedative effects were assessed using a continuous performance test.ResultsNeither alprazolam nor etifoxine affected startle responsivity to U-threat on any of the testing days. While etifoxine reduced the startle response to P-threat on day 1 of treatment for transformed data, a contrary effect of alprazolam was found for raw values. No effects on self-reports and no evidence of sedation could be observed for either drug.ConclusionsNone of the anxiolytic substances had an impact on startle potentiation to U-threat even after several days of intake. The effects of the anxiolytics on startle responsivity to P-threat as well as implications for future studies are discussed.

Effect of nanomolar concentrations of lanthanum on Desmodesmus quadricauda cultivated under environmentally relevant conditions.

Toxicity of lanthanides is generally regarded as low, and they even have been suggested to be beneficial at low concentrations. This research was conducted to investigate effects of Lanthanum (La) on Desmodesmus quadricauda, a freshwater green microalga. The algal cultures were treated with nanomolar La concentrations under controlled environmentally relevant conditions. Intracellular localization of La was analyzed with μXRF tomography in frozen-hydrated samples. At sublethal concentration (128nM) La was in hotspots inside the cells, while at lethal 1387nM that led to release of other ions (K, Zn) from the cells, La filled most of the cells. La had no clear positive effects on growth or photosynthetic parameters, but increasing concentrations led to a dramatic decrease in cell counts. Chlorophyll fluorescence kinetic measurements showed that La led to the inhibition of photosynthesis. Maximal photochemical quantum yield of the PSII reaction center in dark-adapted state (Fv/Fm) decreased at >4.3nM La during the 2nd week of treatment. Minimum dark-adapted fluorescence quantum yield (F0) increased at >13.5nMLa during the 2nd week of treatment except for control (0.2nM La, baseline from chemicals) and 0.3nM La. NPQ at the beginning of the actinic light phase showed significant increase for all the treatments. Metalloproteomics by HPLC-ICPMS showed that La binds to a >500kDa soluble protein complex already in the sub-nM range of La treatments, in the low nM range to a small-sized (3kDa) soluble peptide, and at >100nM La additionally binds to a 1.5kDa ligand.

Dissociation of endocrine responses to the Trier Social Stress Test in Virtual Reality (VR-TSST) by the benzodiazepine alprazolam and the translocator protein 18 kDa (TSPO) ligand etifoxine

BackgroundActivity of the two major stress systems, the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axis, has already been shown to be modulated by different compounds that bind to the central benzodiazepine receptor. Less is known about ligands that modulate the peripheral benzodiazepine receptor – meanwhile known as the translocator protein 18 kDa (TSPO) – which constitute promising candidates in the search of novel anxiolytics. To close this gap, the present study compared the effects of the benzodiazepine alprazolam and the TSPO ligand etifoxine on responses of the HPA and SAM axes to the Trier Social Stress Test, a standardized paradigm to induce acute psychosocial stress in humans, performed in Virtual Reality (VR-TSST). MethodsSixty healthy males, aged between 18 and 55 years, were randomly assigned to receive either a daily dose of 1.5 mg alprazolam, 150 mg etifoxine, or placebo over five days. On the last day of intake, they were exposed to the VR-TSST. We assessed changes of salivary cortisol, allopregnanolone, (nor-) epinephrine in serum, TSPO expression in platelets as well as heart rate (HR), skin conductance level (SCL) and self-reports in response to the stress task. Repeated measures ANOVAs were conducted to examine treatment effects on these stress response variables during the course of VR-TSST. ResultsThe response of salivary cortisol to the VR-TSST was significantly blunted in participants pre-treated with alprazolam but was not affected by etifoxine. While levels of allopregnanolone, epinephrine and norepinephrine increased in response to stress, TSPO expression decreased. None of those endocrine stress markers was affected by the active treatments, whereas TSPO expression increased after etifoxine administration over all study days. There were no effects of the two anxiolytics on HR, SCL or any self-report measurement. ConclusionThe current study confirmed the attenuating effects of benzodiazepines on stress-induced HPA axis activity but did not reveal a comparable effect of the TSPO ligand etifoxine. The long-term consequences of a pharmacologically blunted response of the HPA axis to an acute stressor should be further elucidated. Due to the missing effects of etifoxine on stress-related parameters in our sample of healthy subjects, it might be concluded that the therapeutic effects of this TSPO ligand are restricted to stronger or pathological stress responses, respectively.

Unbinding of Translocator Protein 18 kDa (TSPO) Ligands: From in Vitro Residence Time to in Vivo Efficacy via in Silico Simulations.

Translocator protein 18 kDa (TSPO) is a validated pharmacological target for the development of new treatments for neurological disorders. N,N-Dialkyl-2-phenylindol-3-ylglyoxylamides (PIGAs) are effective TSPO modulators and potentially useful therapeutics for the treatment of anxiety, central nervous system pathologies featuring astrocyte loss, and inflammatory-based neuropathologies. For this class of compounds, no correlation exists between the TSPO binding affinity and the corresponding functional efficacy. Rather, their biological effectiveness correlates with the kinetics of the unbinding events and more specifically with the residence time (RT). So far, the structural reasons for the different recorded RT of congeneric PIGAs remain elusive. Here, to understand the different kinetics of PIGAs, their unbinding paths were studied by employing enhanced-sampling molecular dynamics simulations. Results of these studies revealed how subtle structural differences between PIGAs have a substantial effect on the unbinding energetics. In particular, during the egress from the TSPO binding site, slow-dissociating PIGAs find tight interactions with the protein LP1 region thereby determining a long RT. Further support to these findings was achieved by in vivo studies, which demonstrated how the anxiolytic effect observed for the inspected PIGAs correlated with their RT to TSPO.

Translocator protein 18 kDa ligand alleviates neointimal hyperplasia in the diabetic rat artery injury model via activating PKG

AimsThe proliferation of VSMCs is the pathologic basis for intimal hyperplasia after angioplasty in diabetic patients. Translocator protein (TSPO), located in the outer mitochondrial membrane, has been found to regulate redox intermediate components in cell dysfunction. We hypothesized that TSPO may regulate VSMC proliferation and migration, and be involved in the intimal hyperplasia after angioplasty in diabetes. Materials and methodsCell proliferation was measured by cell counting and MTT assays. Cell migration was measured by Transwell® and scratch-wound assays. TSPO expression in arteries of rats and high glucose-treated A10 cells were detected by immunoblotting and immunofluorescence staining. Neointimal formation of carotid artery was induced by balloon injury in type 2 diabetic rat. Key findingsTSPO expression was increased in the arterial samples from diabetic rats and A10 cells treated with high glucose. Down-regulation of TSPO expression by siRNA decreased the high-glucose-induced VSMC proliferation and migration in A10 cells. This phenomenon could be simulated by using TSPO ligands, PK 11195 and Ro5-4864. cGMP/PKG signals were involved in the TSPO ligand action, since in the presence of cGMP or PKG inhibitor ODQ or KT5823 respectively, the effect of PK 11195 on VSMC proliferation was blocked. Furthermore, PK 11195 significantly inhibited neointimal formation by the inhibition of VSMC proliferation. SignificanceThis study suggests that TSPO inhibition suppresses the proliferation and migration of VSMCs induced by hyperglycemia, consequently, preventing atherosclerosis and restenosis after angioplasty in diabetic conditions. TSPO may be a potential therapeutic target to reduce arterial remodeling induced by angioplasty in diabetes.

Role of Translocator 18 KDa Ligands in the Activation of Leukotriene B4 Activated G-Protein Coupled Receptor and Toll Like Receptor-4 Pathways in Neutrophils.

TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from Escherichia coli (LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4’s actions, as visualized by reductions in L-selectin shedding, β2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites.

Systematic Analysis of Translocator Protein 18 kDa (TSPO) Ligands on Toll-like Receptors-mediated Pro-inflammatory Responses in Microglia and Astrocytes.

Translocator protein 18 kDa (TSPO) is a mitochondrial protein highly expressed on reactive microglia and astrocytes, and is considered as a biomarker for neurodegeneration and brain damage, especially neuroinflammation. Toll-like receptors (TLRs) are closely related with inflammatory responses of microglia and astrocytes and these signaling pathways regulate neuroinflammation. Previous reports have identified the anti-inflammatory effects of TSPO ligands, however study of their effects in relation to the TLR signaling was limited. Here, we investigated the effects of five representative TSPO ligands on microglia and astrocytes following activation by various TLR ligands. Our results show that TSPO ligands reduce the pro-inflammatory response elicited by the TLR ligands with more profound effects on microglia than astrocytes.

Synthesis and in vitro characterization of novel fluorinated derivatives of the translocator protein 18 kDa ligand CfO-DPA-714

The translocator protein 18 kDa (TSPO) is today a validated target for a number of therapeutic applications, but also a well-recognized diagnostic/imaging biomarker for the evaluation of inflammatory related-disease state and progression, prompting the development of specific and dedicated TSPO ligands worldwide. For this purpose, pyrazolo[1,5-a]pyrimidine acetamides constitute a unique class of high affinity and selectivity TSPO ligands; it includes DPA-714, a fluorine-containing derivative that has also been labelled with the positron-emitter fluorine-18, and is nowadays widely used as a Positron Emission Tomography imaging probe. Recently, to prevent defluorination issues encountered in vivo with this tracer, a first series of analogues was reported where the oxygen atom bridging the phenyl ring of the core structure and the fluorinated moiety was replaced with a more robust linkage. Among this new series, CfO-DPA-714 was discovered as a highly promising TSPO ligand. Herein, a novel series of fluorinated analogues of the latter molecule were synthesized and in vitro characterized, where the pharmacomodulation at the amide position of the molecule was explored. Thirteen compounds were thus prepared from a common key-ester intermediate (synthesized in 7 steps from 4-iodobenzoate – 11% overall yield) and a set of commercially available amines and obtained with moderate to good yields (23–81%) and high purities (>95%). With one exception, all derivatives displayed nanomolar to subnanomolar affinity for the TSPO and also high selectivity versus the CBR (Ki (CBR)/Ki (TSPO) > 103). Within this series, three compounds showed better Ki values (0.25, 0.26 and 0.30 nM) than that of DPA-714 (0.91 nM) and CfO-DPA-714 (0.37 nM), and favorable lipophilicity for brain penetration (3.6 < logD7.4 < 4.4). Among these three compounds, the N-methyl-N-propyl amide analogue (9) exhibited similar metabolic stability when compared to CfO-DPA-714 in mouse, rat and human microsomes. Therefore, the latter compound stands out as a promising candidate for drug development or for use as a PET probe, once fluorine-18-labelled, for in vivo neuroinflammation imaging.

Imaging Inflammation in Cerebrovascular Disease.

Imaging inflammation in large intracranial artery pathology may play an important role in the diagnosis of and risk stratification for a variety of cerebrovascular diseases. Looking beyond the lumen has already generated widespread excitement in the stroke community, and the potential to unveil molecular processes in the vessel wall is a natural evolution to develop a more comprehensive understanding of the pathogenesis of diseases, such as ICAD and brain aneurysms.

Novel Pyrazolo[1,5-a]pyrimidines as Translocator Protein 18 kDa (TSPO) Ligands: Synthesis, in Vitro Biological Evaluation, [(18)F]-Labeling, and in Vivo Neuroinflammation PET Images.

A series of novel pyrazolo[1,5-a]pyrimidines, closely related to N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (2, DPA-714), were synthesized and biologically in vitro evaluated for their potential to bind the translocator protein 18 kDa (TSPO), a protein today recognized as an early biomarker of neuroinflammatory processes. This series is composed of fluoroalkyl- and fluoroalkynyl- analogues, prepared from a common iodinated intermediate via Sonogashira coupling reactions. All derivatives displayed subnanomolar affinity for the TSPO (0.37 to 0.86 nM), comparable to that of 2 (0.91 nM). Two of them were radiolabeled with fluorine-18, and their biodistribution was investigated by in vitro autoradiography and positron emission tomography (PET) imaging on a rodent model of neuroinflammation. Brain uptake and local accumulation of both compounds in the AMPA-mediated lesion confirm their potential as in vivo PET-radiotracers. In particular, [(18)F]23 exhibited a significantly higher ipsi- to contralateral ratio at 60 min than the parent molecule [(18)F]2 in vivo.

Synthesis and in vitro characterization of novel fluorinated derivatives of the TSPO 18 kDa ligand SSR180575

SSR180575 (1) is a high-affinity (0.83 nM) TSPO 18 kDa ligand belonging to the pyridazino[4,5-b]indole-1-acetamides family. Herein we describe the synthesis and in vitro characterization of two series of new fluorinated analogues. Eleven compounds (out of fifteen) displayed nanomolar to subnamolar affinities (0.30–8.1 nM) and high selectivities (Ki(CBR)/Ki(TSPO) > 103). Two derivatives stand out as promising candidates for drug development or use as PET probes for in vivo neuroinflammation imaging, once fluorine-18-labelled.

Fate of colloids during estuarine mixing in the Arctic

Abstract. The estuarine behavior of organic carbon (OC) and trace elements (TE) was studied for the largest European sub-Arctic river, which is the Severnaya Dvina; this river has a deltaic estuary covered in ice during several hydrological seasons: summer (July 2010, 2012) and winter (March 2009) baseflow, and the November–December 2011 ice-free period. Colloidal forms of OC and TE were assessed for three pore size cutoffs (1, 10, and 50 kDa) using an in situ dialysis procedure. Conventionally dissolved (&lt; 0.22 μm) fractions demonstrated clear conservative behavior for Li, B, Na, Mg, K, Ca, Sr, Mo, Rb, Cs, and U during the mixing of freshwater with the White Sea; a significant (up to a factor of 10) concentration increase occurs with increases in salinity. Si and OC also displayed conservative behavior but with a pronounced decrease in concentration seawards. Rather conservative behavior, but with much smaller changes in concentration (variation within ±30%) over a full range of salinities, was observed for Ti, Ni, Cr, As, Co, Cu, Ga, Y, and heavy REE. Strong non-conservative behavior with coagulation/removal at low salinities (&lt; 5‰) was exhibited by Fe, Al, Zr, Hf, and light REE. Finally, certain divalent metals exhibited non-conservative behavior with a concentration gain at low (~ 2–5‰, Ba, Mn) or intermediate (~ 10–15‰, Ba, Zn, Pb, Cd) salinities, which is most likely linked to TE desorption from suspended matter or sediment outflux. The most important result of this study is the elucidation of the behavior of the "truly" dissolved low molecular weight LMW&lt; 1 kDa fraction containing Fe, OC, and a number of insoluble elements. The concentration of the LMW fraction either remains constant or increases its relative contribution to the overall dissolved (&lt; 0.22 μm) pool as the salinity increases. Similarly, the relative proportion of colloidal (1 kDa–0.22 μm) pool for the OC and insoluble TE bound to ferric colloids systematically decreased seaward, with the largest decrease occurring at low (&lt; 5‰) salinities. Overall, the observed decrease in the colloidal fraction may be related to the coagulation of organo-ferric colloids at the beginning of the mixing zone and therefore the replacement of the HMW1 kDa–0.22 μm portion by the LMW&lt; 1 kDa fraction. These patterns are highly reproducible across different sampling seasons, suggesting significant enrichment of the mixing zone by the most labile (and potentially bioavailable) fraction of the OC, Fe and insoluble TE. The size fractionation of the colloidal material during estuarine mixing reflects a number of inorganic and biological processes, the relative contribution of which to element speciation varies depending on the hydrological stage and time of year. In particular, LMW&lt; 1 kDa ligand production in the surface horizons of the mixing zone may be linked to heterotrophic mineralization of allochthonous DOM and/or photodestruction. Given the relatively low concentration of particulate versus dissolved load of most trace elements, desorption from the river suspended material was less pronounced than in other rivers in the world. As a result, the majority of dissolved components exhibited either conservative (OC and related elements such as divalent metals) or non-conservative, coagulation-controlled (Fe, Al, and insoluble TE associated with organo-ferric colloids) behavior. The climate warming at high latitudes is likely to intensify the production of LMW&lt; 1 kDa organic ligands and the associated TE; therefore, the delivery of potentially bioavailable trace metal micronutrients from the land to the ocean may increase.

Synthesis of 6-[18F]fluoro-PBR28, a novel radiotracer for imaging the TSPO 18 kDa with PET

6-Fluoro-PBR28 (N-(6-fluoro-4-phenoxypyridin-3-yl)-N-(2-methoxybenzyl)acetamide), a fluorinated analogue of the recently developed TSPO 18 kDa ligand PBR28, was synthesized and labelled with fluorine-18. 6-Fluoro-PBR28 and its 6-chloro/6-bromo counterparts were synthesized in six chemical steps and obtained in 16%, 10% and 19% overall yields, respectively. Labelling with fluorine-18 was performed in one single step (chlorine/bromine-for-fluorine heteroaromatic substitution) using a Zymate-XP robotic system affording HPLC-purified, ready-to-inject, 6-[(18)F]fluoro-PBR28 (>95% radiochemically pure). Non-decay-corrected overall yields were 9-10% and specific radioactivities ranged from 74 to 148 GBq/μmol. In vitro binding experiments, dynamic μPET studies performed in a rat model of acute neuroinflammation (unilaterally, AMPA-induced, striatum-lesioned rats) and ex vivo autoradiography on the same model demonstrated the potential of 6-[(18)F]fluoro-PBR28 to image the TSPO 18 kDa using PET.

Imaging microglial activation during neuroinflammation and Alzheimer's disease.

Microglial activation is an important pathogenic component of neurodegenerative disease processes. This state of increased inflammation is associated not only with neurotoxic consequences but also neuroprotective effects, e.g., phagocytosis and clearance of amyloid in Alzheimer's disease. In addition, activation of microglia appears to be one of the major mechanisms of amyloid clearance following active or passive immunotherapy. Imaging techniques may provide a minimally invasive tool to elucidate the complexities and dynamics of microglial function and dysfunction in aging and neurodegenerative diseases. Imaging microglia in vivo in live subjects by confocal or two/multiphoton microscopy offers the advantage of studying these cells over time in their native environment. Imaging microglia in human subjects by positron emission tomography scanning with translocator protein-18 kDa ligands can offer a measure of the inflammatory process and a means of detecting progression of disease and efficacy of therapeutics over time.

Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: Role of glutamate uptake and FK506 binding protein 12 kDa

Background: The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection.Methods: Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation.Results: When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50–500pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects.Conclusion: The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.

Estrogen receptor α and β subtype expression and transactivation capacity are differentially affected by receptor-, hsp90- and immunophilin-ligands in human breast cancer cells

In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER−) cells stably transfected with either estrogen receptor α (ERα) or β (ERβ) subtype (MDA-MB-231 stably transfected with the mouse ERα cDNA (MERA) and MDA-MB-231 stably transfected with the human ERβ cDNA (HERB), respectively) N-term heat shock protein of 90 kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E 2)-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1 h after E 2 treatment. All hsp90 ligands induced an E 2- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40 kDa (CYP40) interacting in separate ER–hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E 2-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G 1 phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERβ; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.

The p95-100 kDa ligand of the T cell-specific adaptor (TSAd) protein Src-homology-2 (SH2) domain implicated in TSAd nuclear import is p97 Valosin-containing protein (VCP)

T cell-specific adapter protein (TSAd) is required for normal T cell antigen receptor (TCR)-induced transcription of cytokine genes in T cells. How TSAd controls cytokine transcription is unknown. Previously, we have shown that TSAd is actively transported to the nucleus of T cells suggesting that this adapter may in part function within this cellular compartment. Nuclear translocation of TSAd is dependent upon an intact Src-homology-2 (SH2) domain and a p95-100 kDa ligand of the SH2 domain has been implicated in nuclear import. Here, using microchemical techniques, we identify p95-100 as p97 Valosin-containing protein (VCP) whose homolog in yeast is the cell division control protein, CDC48. Physical interaction between TSAd and VCP can be demonstrated between endogenous proteins in T cells. Interaction is direct and is dependent upon phosphorylation of tyrosine residue 805 of VCP that has been previously recognized as a major target of tyrosine kinase(s) involved in TCR signaling. Significantly, with the use of CDC48 mutant yeast, we demonstrate that VCP/CDC48 is required for transport of TSAd into the eukaryotic nucleus. These findings provide important insights into the mechanism of TSAd nuclear import and the role of TSAd in T cell signal transduction.

The role of chaperone proteins in the aryl hydrocarbon receptor core complex

The aryl hydrocarbon receptor (AhR) exists in the absence of a ligand as a tetrameric complex composed of a 95–105 kDa ligand binding subunit, a dimer of hsp90, and the immunophilin-like X-associated protein 2 (XAP2). XAP2 has a highly conserved carboxy terminal tetratricopeptide repeat domain that is required for both hsp90 and AhR binding. Hsp 90 appears to be involved in the initial folding of newly synthesized AhR, stabilization of ligand binding conformation of the receptor, and inhibition of constitutive dimerization with ARNT. XAP2 is capable of stabilizing the AhR, as well as enhancing cytoplasmic localization of the receptor. XAP2 binds to both the AhR and hsp90 in the receptor complex, and is capable of independently binding to both hsp90 and the AhR. However, the exact functional role for XAP2 in the AhR complex remains to be fully established.

Signaling mediated by PrPc interaction with laminin and the 66 KDa ligand protein

Signaling mediated by PrPc interaction with laminin and the 66 KDa ligand protein