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High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

ABSTRACT: High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

PROVIDER: PRJNA612056 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11292334_1.fastq.gz	Fastqsanger.gz
	SRR11292334_2.fastq.gz	Fastqsanger.gz
	SRR11292335_1.fastq.gz	Fastqsanger.gz
	SRR11292335_2.fastq.gz	Fastqsanger.gz
	SRR11292336_1.fastq.gz	Fastqsanger.gz

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High Throughput pMHC-I Tetramer Library Production Using Chaperone Mediated Peptide Exchange

Project description:Peptide exchange technologies are essential for the generation of pMHC-multimer libraries for probing diverse, polyclonal TCR repertoires in various settings. Here we report, using the molecular chaperone TAPBPR, a robust method for the capture of stable, empty MHC-I molecules, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange ‘goldilocks’ peptides with high-affinity peptides of interest in an overnight reaction. Using the same system, we describe high-throughput assays to validate binding of multiple candidate peptides on the empty MHCI groove. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment.

2020-03-12 | GSE146826 | GEO

High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange.

Project description:Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.

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A replisome-associated parental histone H3-H4 tetramer chaperone required for faithful epigenetic inheritance

Project description:Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel relatively large distances from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

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A replisome-associated parental histone H3-H4 tetramer chaperone required for faithful epigenetic inheritance

Project description:A replisome-associated parental histone H3-H4 tetramer chaperone required for faithful epigenetic inheritance

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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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Regulators of histone exchange

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TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome

Project description:THOC6 is the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants do not alter the expression of TREX dimer component proteins in human cells, but reduced binding affinity to ALYREF implicates impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for regulation of key signaling pathways in human corticogenesis that dictate the transition from proliferative to neurogenic divisions, developmental biology implicated in TIDS neuropathology.

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