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71.
Florian G?rtner Thorsten Seidel Uwe Schulz Jan Gummert Hendrik Milting 《The Journal of biological chemistry》2013,288(45):32138-32148
Endothelin receptor A (ETA), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ETA mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ETA desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gαq coupling-deficient mutant GRK2-D110A suppressed ETA-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ETA-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ETA-WT and ETA-6PD. This demonstrates that ETA desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gαq and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ETA-WT and ETA-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ETA-WT and the phosphorylation-deficient mutant ETA-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism. 相似文献
72.
Sonja Sucic Florian Koban Ali El-Kasaby Oliver Kudlacek Thomas Stockner Harald H. Sitte Michael Freissmuth 《The Journal of biological chemistry》2013,288(8):5330-5341
The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif (607RI608 in SERT). Substituting Lys610 with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters. 相似文献
73.
Birte Plitzko Gudrun Ott Debora Reichmann Colin J. Henderson C. Roland Wolf Ralf Mendel Florian Bittner Bernd Clement Antje Havemeyer 《The Journal of biological chemistry》2013,288(28):20228-20237
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. 相似文献
74.
Carmen F. Ludwig Florian Ullrich Lilia Leisle Tobias Stauber Thomas J. Jentsch 《The Journal of biological chemistry》2013,288(40):28611-28619
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. 相似文献
75.
Taeko Kimura Koji Tsutsumi Masato Taoka Taro Saito Masami Masuda-Suzukake Koichi Ishiguro Florian Plattner Takafumi Uchida Toshiaki Isobe Masato Hasegawa Shin-ichi Hisanaga 《The Journal of biological chemistry》2013,288(11):7968-7977
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. 相似文献
76.
77.
Bernat Elvira Sabina Honisch Ahmad Almilaji Tatsiana PakladokGuilai Liu Ekaterina ShumilinaIoana Alesutan Wenting YangCarlos Munoz Florian Lang 《生物化学与生物物理学报:生物膜》2013
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 (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). 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 Ig, 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. 相似文献
78.
Ingo V. Hartung Marion Hitchcock Florian Pühler Roland Neuhaus Arne Scholz Stefanie Hammer Kirstin Petersen Gerhard Siemeister Dominic Brittain Roman C. Hillig 《Bioorganic & medicinal chemistry letters》2013,23(8):2384-2390
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. 相似文献
79.
Florian Hubrich Silja Mordhorst Jennifer N. Andexer 《Bioorganic & medicinal chemistry letters》2013,23(5):1477-1481
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 Km 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. 相似文献
80.