Sites on TAPBPR that scaffold assembly of a MHC-I H chain with beta2m are important for TAPBPR chaperone activity
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PROVIDER: PRJNA613093 | ENA |
REPOSITORIES: ENA
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Project description:TAP-binding protein-related (TAPBPR) facilitates the processing of nascent class I MHC (MHC-I) by (1, chaperone function) acting as a chaperone for misfolded or partially folded MHC-I substrates in the cell and (2, editing function) by catalyzing exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. In cells in which the principal MHC-I-specific chaperone and TAPBPR homologue tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of TAPBPR can partially replace tapasin and rescue HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, TAPBPR was deep mutationally scanned at 92 positions in the core, at the interface with MHC-I, and on the ‘backside’ distal from where MHC-I binds. Critical regions of TAPBPR for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain and residues that contact the junction between beta-2 microglobulin and alpha-3 domains. Key sites on TAPBPR for chaperone activity therefore scaffold assembly of the MHC-I H chain with beta-2 microglobulin.
2020-03-18 | GSE147137 | GEO
Project description:TAP-binding protein-related (TAPBPR) is an endoplasmic reticulum-resident chaperone that facilitates class I MHC (MHC-I) processing and peptide loading. TAPBPR has (1) chaperone function to stabilize misfolded or partially folded nascent MHC-I substrates, and (2) editing function, in which it catalyzes the exchange of low affinity for high affinity antigenic peptides in the MHC-I peptide-binding groove. TAPBPR-TM is a chimera of the TAPBPR ectodomain and a canonical TM domain, which escapes the endoplasmic reticulum to reach the cell surface. We reasoned that at the cell surface, TAPBPR-TM is more likely to interact with folded MHC-I and thus surface interactions will be more representative of editing function. When tapasin, a homolog of TAPBPR and the main MHC-I-specific chaperone, is knocked out, surface trafficking of HLA-A2 (a human MHC-I allele) is severely diminished, but surface HLA-A2 levels are rescued by over-expression of TAPBPR-TM. Using this assay as the foundation for a fluorescence-based selection, we deep mutationally scanned 104 positions on TAPBPR-TM to identify sites critical for engaging folded MHC-I.
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Project description:Tapasin acts as the principal MHC-I-specific chaperone for facilitating folding and antigenic peptide loading of nascent MHC class I substrates in the cell. In cells where tapasin has been knocked out, the processing and surface trafficking of the human MHC-I allele HLA-A2 is substantially reduced. Over-expression of tapasin rescues HLA-A2 surface expression. Using this assay as the basis for a fluorescence-based selection, tapasin was deep mutationally scanned at 108 positions in the core, at the interface with MHC-I, and on the ‘backside’ distal from where MHC-I binds. Critical residues of tapasin for rescue of HLA-A2 processing map to sites that contact the underside of the MHC-I alpha-2 domain, to the surface contacting the MHC-1 beta2m and alpha-3 domains, and to the base of a protruding loop that rests above the peptide-binding groove (but not to the tip of the loop itself).
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Project description:A deep mutational scan of the MHC class I-specific chaperone tapasin
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Project description:This study investigated early host reactions to implanted materials to predict successful tissue regeneration with implant. Three kinds of scaffold, i.e., non-coat, collagen-coated, and PMB-coated porous polystylene scaffolds were implanted subcutaneously in mice dorsal area. Those scaffolds were used as bio-incomopatible materials, appropriate materials for tissue regeneration (bio active), and inappropriate to regenration (bio-inert) scaffolds. Seven days after implantation, scaffolds were explanted and total RNA was isolated from infiltrated host cells into scaffold by laser microdissection. Gene expressions of cells in collagen- and PMB-coated scaffold were normalized using results of non coat scaffold. Genes with more than 2-fold difference between collagen and PMB were picked up and narrowed to related keywords; inflammation, angiogenesis, wound healing, and mcrophage polarization. Among those genes, interluekin (IL)-1beta which promote both inflammation and wound healing was up-regulated in collagen-coated scaffold. On the other hand, IL-10 which suppress both inflammation and wound healing was up-regulated in PMB-coated scaffold. Angiogenesis-promoting genes were up-regulated and angiogenesis suppressve genes were suppressed in collagen. Up-regulation of IL-1b and the angiogenesis-relating genes inside the porous scaffolds are the possibly important factors for controlling tissue regeneration. Three-condition experiment, host cells infiltrated in non coat (reference), collagen-coated, and PMB-coated scaffolds. Two-microarray condition experiments, collagen vs. non coat and PMB coat vs. non coat. Hybridization: 2 replicates. Scanning: 3 replicates. Biological experiments: once.
2014-01-23 | E-GEOD-52053 | biostudies-arrayexpress
Project description:At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (beta2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the alpha-3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the alpha-3 domain of one FHC binds to another FHC in a manner similar to the beta2m light chain.
2022-05-11 | PXD033485 | Pride
Project description:The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). NMR experiments reveal that conformational dynamics of specific MHC-I allotypes, especially around regions known to be in physical proximity to the TAPBPR interface, are correlated with TAPBPR binding affinity. HLA-A*02:01 residues in the alpha1 and alpha2 domains were deep mutationally scanned to determine the effects of nearly all single amino acid substitutions on HLA-A2 surface expression and TAPBPR interactions. Surface trafficking, which demands HLA-A2 be properly folded with bound peptide, imposes strict sequence constraints on the HLA-A2 core, surfaces contacting the beta2-microglobulin and alpha3 domains, and on the A, B and F pockets that ‘grip’ peptide anchor residues. However, TAPBPR binding is permissive to many mutations of HLA-A2, both in the core and on the surface, with the exceptions of two conserved clusters of hydrophobic residues that pack the alpha2-1 and alpha2-2 helices against the beta-sheet. TAPBPR binding is therefore dependent on a local conformation of the alpha2 domain, most likely with a widened peptide binding groove based on published crystal structures. Overall, the data are consistent with a conformational selection model for MHC-I/TAPBPR interactions, in which high MHC-I dynamics increases representation in the structural ensemble of appropriate conformers for TAPBPR recognition.
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Project description:Global microarray (HG U133 Plus 2.0) was used for the first time to investigate the effects of resistance exercise on the transcriptome in slow-twitch myosin heavy chain (MHC) I and fast-twitch MHC IIa muscle fibers of young and old women. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program. A total of 14 females were included in this investigation. The participants included 8 young (23±2y) and 6 old (85±1y) females. All subjects participated in 12 wks of progressive resistance training consisting of bilateral knee extensions with 3x10 reps at 70% of 1-RM, and 3d/wk for a total of 36 training sessions. Vastus lateralis biopsies were obtained in conjunction with the 1st and 36th (last) training session and included a basal biopsy and another biopsy 4hrs post the resistance exercise session. From each biopsy sample, we isolated individual muscle fibers. After myosin isoform identification of isolated fibers (SDS-PAGE), RNA extraction of 20 MHC I and 20 MHC IIa muscle fibers per biopsy sample followed. Thus, each resulting sample contained total RNA from 20 muscle fibers of identical fiber type (MHC I or MHC IIa). A total of 70 samples were analyzed on separate microarray chips, and samples were not pooled between subjects. The study design allowed us to examine the acute effects of resistance exercise on the transcriptome in MHC I and MHC IIa muscle fibers in the untrained and trained state.
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Project description:Global microarray (HG U133 Plus 2.0) was used for the first time to investigate the effects of resistance exercise on the transcriptome in slow-twitch myosin heavy chain (MHC) I and fast-twitch MHC IIa muscle fibers of young and old women. Vastus lateralis muscle biopsies were obtained pre and 4hrs post resistance exercise in the beginning (untrained state) and at the end (trained state) of a 12 wk progressive resistance training program.
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