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  • Article
    Herget M, Kreissig N, Kolbe C, Schölz C, Tampé R, Abele R.
    J Biol Chem. 2009 Dec 04;284(49):33740-9.
    The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.
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  • Article
    San José E, Alarcón B.
    J Biol Chem. 1999 Nov 19;274(47):33740-6.
    In the absence of ligand, the T cell receptor (TCR)/CD3 complex is continuously internalized and recycled to the cell surface, whereas receptor engagement results in its down-regulation. The present study shows that the TCR and CD3 components follow different fates accompanying their constitutive internalization. Although the CD3 moiety is recycled to the cell surface, the TCR heterodimer is degraded and replaced by newly synthesized chains. Since the TCR heterodimer cannot reach the cell membrane on its own, we propose a model in which recycling CD3 is transported along a retrograde pathway to the endoplasmic reticulum, where it associates with newly made TCR. Interestingly, engagement of the TCR.CD3 complex by superantigen resulted not only in the down-regulation of the TCR and CD3 components but also caused a transient stabilization of the TCR heterodimer. This suggests that TCR engagement diverts the TCR heterodimer from a degradation to a recycling pathway. Contrary to CD3, the intracellular fate of the TCR heterodimer is thus regulated, providing a mechanism for rapidly replacing nonfunctional TCR during intrathymic development of T cells.
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  • Article
    Roder K, Werdich AA, Li W, Liu M, Kim TY, Organ-Darling LE, Moshal KS, Hwang JM, Lu Y, Choi BR, MacRae CA, Koren G.
    J Biol Chem. 2014 Dec 05;289(49):33730-40.
    Two recent studies (Newton-Cheh, C. et al. (2009) Common variants at ten loci influence QT interval duration in the QTGEN Study. Nat. Genet. 41, 399-406 and Pfeufer, A. et al. (2009) Common variants at ten loci modulate the QT interval duration in the QTSCD Study. Nat. Genet. 41, 407-414) identified an association, with genome-wide significance, between a single nucleotide polymorphism within the gene encoding RING finger protein 207 (RNF207) and the QT interval. We sought to determine the role of RNF207 in cardiac electrophysiology. Morpholino knockdown of RNF207 in zebrafish embryos resulted in action potential duration prolongation, occasionally a 2:1 atrioventricular block, and slowing of conduction velocity. Conversely, neonatal rabbit cardiomyocytes infected with RNF207-expressing adenovirus exhibited shortened action potential duration. Using transfections of U-2 OS and HEK293 cells, Western blot analysis and immunocytochemistry data demonstrate that RNF207 and the human ether-a-go-go-related gene (HERG) potassium channel interact and colocalize. Furthermore, RNF207 overexpression significantly elevated total and membrane HERG protein and HERG-encoded current density by ∼30-50%, which was dependent on the intact N-terminal RING domain of RNF207. Finally, coexpression of RNF207 and HSP70 increased HERG expression compared with HSP70 alone. This effect was dependent on the C terminus of RNF207. Taken together, the evidence is strong that RNF207 is an important regulator of action potential duration, likely via effects on HERG trafficking and localization in a heat shock protein-dependent manner.
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  • Article
    Kim YS, Han CY, Kim SW, Kim JH, Lee SK, Jung DJ, Park SY, Kang H, Choi HS, Lee JW, Pak YK.
    J Biol Chem. 2001 Sep 07;276(36):33736-40.
    Small heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but was suppressed by oxidized low density lipoprotein (oxLDL) and its constituent 13-hydroxyoctadecadienoic acid, a ligand for peroxisome proliferator-activated receptor gamma. Furthermore, SHP acted as a transcription coactivator of nuclear factor-kappa B (NF kappa B) and was essential for the previously described NF kappa B transactivation by palmitoyl lysophosphatidylcholine, one of the oxLDL constituents. Accordingly NF kappa B, which was transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF kappa B in oxLDL-treated, resting macrophage cells.
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  • Article
    Fann MC, Maloney PC.
    J Biol Chem. 1998 Dec 11;273(50):33735-40.
    UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.
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