The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Organophosphate poisoning in the developed world – A single centre experience from here to the millennium

Organophosphate (OP) poisoning is still associated with high morbidity and mortality rates, both in resource-poor settings and in well-developed countries. Despite numerous publications dealing with this particular poison, detailed clinical data on more severe overdoses with these agents are relatively sparsely reported. A retrospective study was consequently conducted on 33 patients with OP poisoning admitted to our intensive care unit (ICU) to provide additional data on clinical features. We included moderate to severe poisonings between 2000 and 2012 who required admission to ICU.     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed—despite being orally bioavailable and not a P-glycoprotein substrate—much lower brain/plasma exposure ratios than PD325901.     

Cinnamic acid derivatives as inhibitors for chorismatases and isochorismatases

Chorismatases and isochorismatases catalyse the hydrolysis of chorismate or isochorismate leading to unsaturated cyclohexenoic acid derivatives. Based on simplification of the physiological substrates, two cinnamic acid-derived compounds, differing in the saturation of the side chain, were developed. In contrast to earlier inhibitor studies, the compounds described here do not have an ether bond and therefore can be synthesised very easily in one or two steps without the need for protective groups. Both substances demonstrate inhibition of the isochorismatase EntB from Escherichia coli and the chorismatases FkbO and Hyg5 from Streptomyces. For chorismatases, the unsaturated compound shows IC₅₀ values in the millimolar range, while the saturated compound is the better inhibitor with IC₅₀ values in the micromolar/low millimolar range; for the isochorismatase tested both compounds inhibit in the micromolar range. Further, an analysis of the apparent K_m values for FkbO and EntB was performed, showing that both inhibitors act in a competitive manner. Due to the ease of modifying these new inhibitors they are a suitable starting point for exploring further functionalised derivatives.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.