Probing binding and occlusion of substrate in the human creatine transporter-1 by computation and mutagenesis.

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane-embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148-creatine-Na+ is essential to the process of occlusion, which occurs via a "hold-and-pull" mechanism. The model should be useful to study the impact of disease-associated point mutations on the folding of CRT1 and identify approaches which correct folding-deficient mutants.

Plant-Derived Cyclotides Modulate κ-Opioid Receptor Signaling.

Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low µM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A1-13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

The Creatine Transporter Unfolded: A Knotty Premise in the Cerebral Creatine Deficiency Syndrome.

Creatine provides cells with high-energy phosphates for the rapid reconstitution of hydrolyzed adenosine triphosphate. The eponymous creatine transporter (CRT1/SLC6A8) belongs to a family of solute carrier 6 (SLC6) proteins. The key role of CRT1 is to translocate creatine across tissue barriers and into target cells, such as neurons and myocytes. Individuals harboring mutations in the coding sequence of the human CRT1 gene develop creatine transporter deficiency (CTD), one of the pivotal underlying causes of cerebral creatine deficiency syndrome. CTD encompasses an array of clinical manifestations, including severe intellectual disability, epilepsy, autism, development delay, and motor dysfunction. CTD is characterized by the absence of cerebral creatine, which implies an indispensable role for CRT1 in supplying the brain cells with creatine. CTD-associated variants dramatically reduce or abolish creatine transport activity by CRT1. Many of these are point mutations that are known to trigger folding defects, leading to the retention of encoded CRT1 proteins in the endoplasmic reticulum and precluding their delivery to the cell surface. Misfolding of several related SLC6 transporters also gives rise to detrimental pathologic conditions in people; e.g., mutations in the dopamine transporter induce infantile parkinsonism/dystonia, while mutations in the GABA transporter 1 cause treatment-resistant epilepsy. In some cases, folding defects are amenable to rescue by small molecules, known as pharmacological and chemical chaperones, which restore the cell surface expression and transport activity of the previously non-functional proteins. Insights from the recent molecular, animal and human case studies of CTD add toward our understanding of this complex disorder and reveal the wide-ranging effects elicited upon CRT1 dysfunction. This grants novel therapeutic prospects for the treatment of patients afflicted with CTD, e.g., modifying the creatine molecule to facilitate CRT1-independent entry into brain cells, or correcting folding-deficient and loss-of-function CTD variants using pharmacochaperones and/or allosteric modulators. The latter justifies a search for additional compounds with a capacity to correct mutation-specific defects.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson's Disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation.

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials. KEY MESSAGES: Pretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs. Vildagliptin enhances in vitro migration of pretreated HSPCs. Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs. Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.

Nature-Derived Peptides: A Growing Niche for GPCR Ligand Discovery.

G protein-coupled receptors (GPCRs) represent important drug targets, as they regulate pivotal physiological processes and they have proved to be readily druggable. Natural products have been and continue to be amongst the most valuable sources for drug discovery and development. Here, we surveyed small molecules and (poly-)peptides derived from plants, animals, fungi, and bacteria, which modulate GPCR signaling. Among naturally occurring compounds, peptides from plants, cone-snails, snakes, spiders, scorpions, fungi, and bacteria are of particular interest as lead compounds for the development of GPCR ligands, since they cover a chemical space, which differs from that of synthetic small molecules. Peptides, however, face challenges, some of which can be overcome by studying plant-derived compounds. We argue here that the opportunities outweigh the challenges.

Trafficking of the amino acid transporter B0,+ (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum.

A plasma membrane amino acid transporter B0,+ (ATB0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB0,+ trafficking. Rat ATB0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export "RI" motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.

Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome.

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS. KEY MESSAGES: Murine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors. Treprostinil reduces busulfan-induced cell death in vitro. Treprostinil lowers liver weight-to-body weight ratios in mice. Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.

Identification and characterization of the Fasciola hepatica sodium- and chloride-dependent taurine transporter.

The parasitic liver fluke Fasciola hepatica infests mainly ruminants, but it can also cause fasciolosis in people, who ingest the metacercariae encysted on plants. The drug of choice to treat fasciolosis is triclabendazole (TBZ), which has been on the market for several decades. This is also true for the other available drugs. Accordingly, drug-resistant flukes have been emerging at an increasing rate making it desirable to identify alternative drug targets. Here, we focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. A common way to render bile acids less toxic is to conjugate them to taurine (2-aminoethanesulfonic acid). We cloned a transporter from the solute carrier-6 (SLC6) family, which was most closely related to the GABA-transporter-2 of other organisms. When heterologously expressed, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM = 12.0 ± 0.5 µM) but not of GABA. Substrate uptake was dependent on Na+- and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 = 14±3 mM) than the human taurine transporter (EC50 = 55±7 mM). We incubated flukes with unconjugated bile acids in the presence and absence of taurine: taurine promoted survival of flukes; the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. Based on these observations, we conclude that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a candidate drug target.

Hyponatremia and V2 vasopressin receptor upregulation: a result of HSP90 inhibition.

PURPOSE: Small-molecule inhibitors of heat-shock protein 90 (HSP90) have been under development as chemotherapeutic agents. The adverse events reported from early clinical trials included hyponatremia. Given the limited number of patients enrolled, the number of hyponatremia incidents was remarkable and repeatedly, the event was judged as severe. Inappropriate V2 vasopressin receptor stimulation is an established cause of hyponatremia. We explored the hypothesis that HSP90 inhibition produces hypersensitivity to vasopressin by upregulating V2-receptors. METHODS: Experiments were carried out in cell culture using HEK293 cells with heterologous expression of the human V2-receptor and HELA cells with an endogenous V2-receptor complement. We tested the effect of HSP90 inhibition by three structurally unrelated compounds (alvespimycin, luminespib, radicicol) and asserted its specificity in cells depleted of cytosolic HSP90 (by RNA interference). Assays encompassed surface V2-receptor density and vasopressin-stimulated formation of cyclic AMP (cAMP). RESULTS: The results demonstrate a twofold increase in cell-surface receptor density following pre-incubation with each of the HSP90 inhibitors. The effect had a concentration-dependence consistent with the individual potencies to inhibit HSP90. Similarly, depletion of cytosolic HSP90 increased surface-receptor density and at the same time, reduced the inhibitor effect. Upregulated V2-receptors were fully functional; hence, in culture treated with an HSP90 inhibitor, addition of vasopressin resulted in higher levels of cAMP than in controls. CONCLUSION: Since formation of cAMP is the first signalling step in raising water permeability of the collecting duct epithelia, we suggest that V2-receptor upregulation generates hypersensitivity to vasopressin linking HSP90 inhibition to the development of hyponatremia.

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Comparison of the ß-Adrenergic Receptor Antagonists Landiolol and Esmolol: Receptor Selectivity, Partial Agonism, and Pharmacochaperoning Actions.

Blockage of ß1-adrenergic receptors is one of the most effective treatments in cardiovascular medicine. Esmolol was introduced some three decades ago as a short-acting ß1-selective antagonist. Landiolol is a more recent addition. Here we compared the two compounds for their selectivity for ß1-adrenergic receptors over ß2-adrenergic receptors, partial agonistic activity, signaling bias, and pharmacochaperoning action by using human embryonic kidney (HEK)293 cell lines, which heterologously express each human receptor subtype. The affinity of landiolol for ß1-adrenergic receptors and ß2-adrenergic receptors was higher and lower than that of esmolol, respectively, resulting in an improved selectivity (216-fold versus 30-fold). The principal metabolite of landiolol (M1) was also ß1-selective, but its affinity was very low. Both landiolol and esmolol caused a very modest rise in cAMP levels but a robust increase in the phosphorylation of extracellular signal regulated kinases 1 and 2, indicating that the two drugs exerted partial agonist activity with a signaling bias. If cells were incubated for ≥24 hours in the presence of ≥1 µM esmolol, the levels of ß1-adrenergic-but not of ß2-adrenergic-receptors increased. This effect was contingent on export of the ß1-receptor from endoplasmic reticulum and was not seen in the presence of landiolol. On the basis of these observations, we conclude that landiolol offers the advantage of: 1) improved selectivity and 2) the absence of pharmacochaperoning activity, which sensitizes cells to rebound effects upon drug discontinuation.

Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation.

Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [(3)H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg(-1) 8 h(-1)) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application.

STAT3 controls IL6-dependent regulation of serotonin transporter function and depression-like behavior.

Experimental evidence suggests a role for the immune system in the pathophysiology of depression. A specific involvement of the proinflammatory cytokine interleukin 6 (IL6) in both, patients suffering from the disease and pertinent animal models, has been proposed. However, it is not clear how IL6 impinges on neurotransmission and thus contributes to depression. Here we tested the hypothesis that IL6-induced modulation of serotonergic neurotransmission through the STAT3 signaling pathway contributes to the role of IL6 in depression. Addition of IL6 to JAR cells, endogenously expressing SERT, reduced SERT activity and downregulated SERT mRNA and protein levels. Similarly, SERT expression was reduced upon IL6 treatment in the mouse hippocampus. Conversely, hippocampal tissue of IL6-KO mice contained elevated levels of SERT and IL6-KO mice displayed a reduction in depression-like behavior and blunted response to acute antidepressant treatment. STAT3 IL6-dependently associated with the SERT promoter and inhibition of STAT3 blocked the effect of IL6 in-vitro and modulated depression-like behavior in-vivo. These observations demonstrate that IL6 directly controls SERT levels and consequently serotonin reuptake and identify STAT3-dependent regulation of SERT as conceivable neurobiological substrate for the involvement of IL6 in depression.

Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.

Perioperative kinetics of parathyroid hormone in unilateral, primary thyroid surgery.

Dysfunction of the parathyroid glands is an important cause of complications after thyroid surgery. Intraoperative monitoring of the function of the parathyroid glands can be performed using parathyroid hormone (PTH) kinetics. Unilateral thyroid surgery is associated with a decreased risk for postoperative hypocalcemia (POH) and permanent hypoparathyroidism (PEH). We focused on unilateral thyroid surgery by monitoring the functionality of the parathyroid glands and comparing the perioperative PTH kinetics of patients with and without POH. In a prospective study, 143 patients scheduled for unilateral thyroid surgery underwent monitoring of perioperative changes in serum PTH and serum calcium levels, and of clinical symptoms of hypocalcemia. The rates of POH and PEH were 18.2 and 0%, respectively. In patients without POH, PTH significantly increased from the time of skin incision to the end of the operation and after the operation (20.1 pg/ml, IQR 15.5-26.8 vs. 21.4 pg/ml, IQR 16.4-29.5; p=0.005), which was not the case in patients who developed POH. In a multivariate analysis of predictive factors for POH, two parameters became significant, namely female gender (odds ratio 6.87, 95% confidence interval 0.92-51.01) and lower initial serum calcium levels (odds ratio 3.54*e(-8), 95% confidence interval 3.63*e(-12); 0.00). The rate of POH was unexpectedly high. Rather than intraoperative PTH declines, an unstable balance of factors that influence calcium metabolism likely is the major contributor to POH after unilateral thyroid surgery. There was no case of PEH after unilateral, primary thyroid surgery, which underlines the need for an individualized approach to the extent of resection.

A cytosolic relay of heat shock proteins HSP70-1A and HSP90ß monitors the folding trajectory of the serotonin transporter.

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90ß. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90ß by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90ß. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.