ABCB9 p.Lys545Ala
| Predicted by SNAP2: | A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (91%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Identification of a lysosomal peptide transport sy... J Biol Chem. 2007 Dec 28;282(52):37836-43. Epub 2007 Oct 31. Demirel O, Waibler Z, Kalinke U, Grunebach F, Appel S, Brossart P, Hasilik A, Tampe R, Abele R
Identification of a lysosomal peptide transport system induced during dendritic cell development.
J Biol Chem. 2007 Dec 28;282(52):37836-43. Epub 2007 Oct 31., [PMID:17977821]
Abstract [show]
The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
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47 TAPL-K545A/ H699A was generated by Ligase Chain Reaction using the primers 5Ј-CGGGCAGTGGGGCGAGCTCCTGTGTC-3Ј and 5Ј-CTC- ATCATCGCGGCCCGGCTGAGCAC-3Ј (mutated bases are underlined).
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ABCB9 p.Lys545Ala 17977821:47:5
status: NEW75 Retroviral Transduction-Recombinant retroviruses were obtained by co-transfection of pCL-Ampho (generous gift of Peter Cresswell) (28), and the retroviral constructs pLPCX- TAPL or pLPCX-TAPL-K545A/H699A into 293T cells with Lipofectamine 2000 (Invitrogen) following the manufacturer`s instructions.
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ABCB9 p.Lys545Ala 17977821:75:192
status: NEW198 After retroviral transduction, TAPL and the Walker A/H-loop mutant K545A/H699A were detected by immunoblotting as single bands (Fig. 4A).
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ABCB9 p.Lys545Ala 17977821:198:67
status: NEW213 Perinuclear supernatants (PNS) derivedfromTAPL-wt-orTAPL-K545A/H699A-transducedRajicellswerefrac- tioned by a Percoll gradient and analyzed by SDS-PAGE and immunoblotting.
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ABCB9 p.Lys545Ala 17977821:213:57
status: NEW