Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Digestion of protein substrates by 20S proteasomes

ABSTRACT: The proteasome is the central protease in generating peptide repertoires for antigen presentation on human leukocyte antigen class I (HLA-I). Proteasomes not only can catalyze peptide hydrolysis but are also capable of mediating transpeptidation, resulting in spliced peptides generation, a process known as proteasome-catalyzed peptide splicing (PCPS). Despite its immunological importance, it is still unclear what are the determinants of PCPS that favor transpeptidation over hydrolysis. Hence, we sought to characterize the processing of many protein substrates and their peptide products by using a simplistic in vitro antigen processing system and analyzed by mass spectrometry followed by a refined downstream data analysis pipeline.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Michele Mishto Dr. Juliane Liepe

PROVIDER: MSV000092631 | MassIVE | Wed Aug 09 01:39:00 BST 2023

SECONDARY ACCESSION(S): PXD044463

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Similar Datasets

20S proteasome digestion of HeLa cell extracts

Project description:The 20S proteasome is responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. Here we conducted a comprehensive analysis of the 20S CP substrates in-vitro. We revealed that the 20S CP substrates are highly structually disordered. The 20S proteasome substrate group, termed 20S-IDPome are characterized by having significantly more protein binding partners, more post-translational modification sites and are highly enriched for RNA binding proteins. Remarkably, we found that low complexity proteins with prion-like domain which interact with GR or PR di-peptide repeats are the most preferential 20S proteasome substrates. Our finding suggests roles of the 20S proteasome in gene transcription and formation of phase-separated granules.

2018-07-30 | PXD010132 | Pride

KRAS G12V digestion by 20S proteasome

Project description:KRAS is a well known tumor associated antigen. It’s a GTPase that functions as molecular switch in regulatory pathways responsible for proliferation and survival. It’s frequently mutated in a variety of cancers. One of the possible driver mutations is G12V substitution, which impairs KRAS GTPase activity and renders the mutants persistently in the GTP-bound active form, thereby promoting tumorigenesis and tumor malignancy. Its high frequency in cancers makes KRAS an attractive target for immunotherapy. We performed in vitro digestions of synthetic polypeptide corresponding to KRAS2-35 carrying the mutation with purified 20S proteasome. Samples were measured by LC-MS/MS.

2019-10-24 | PXD015580 | Pride

Elucidation of the Signatures of Proteasome-Catalyzed Peptide Splicing

Project description:Proteasomes catalyze the degradation of endogenous proteins into oligopeptides, but can concurrently create spliced oligopeptides through ligation of previously non-contiguous peptide fragments. Recent studies have uncovered a formerly unappreciated role for proteasome-catalyzed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound cis-spliced peptides that can be targeted by CD8+ T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalyzed cis-splicing reactions by examination of in vitro proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based de novo sequencing workflow. We show that forward and reverse PCPS reactions display unique splicing signatures, defined by preferential fusion of distinct amino acid residues with stringent peptide length distributions, suggesting sequence- and size-dependent accessibility of splice reactants for proteasomal substrate binding pockets. Our data provide the basis for a more informed mechanistic understanding of PCPS

2021-05-17 | PXD025893 | Pride

Digestion of TSN2 and TSN89 synthetic peptides by proteasomes

Project description:Proteasomes are large multi-subunit enzymes that act as the main producers of antigenic peptides presented at the cell surface to CD8+ T cells. They can simply cut proteins or ligate non-contiguous peptide fragments thereby generating novel sequences, i.e. spliced peptides via a process called proteasome-catalysed peptide splicing (PCPS). In order to benchmark methods for spliced peptide identification, we here perform in vitro digestions of TSN2 and TSN89 synthetic polypeptide substrates by K562-derived 20S proteasome followed by LC-MS/MS measurements using Orbitrap Fusion Lumos mass spectrometer. Michele Mishto, Head of the research group Molecular Immunology at King’s College London and the Francis Crick Institute, London (UK). Email: michele.mishto@kcl.ac.uk

2022-12-19 | PXD025995 | Pride

Digestion of a variety of synthetic peptides by proteasomes

Project description:Proteasomes are large multi-subunit enzymes that act as the main producers of antigenic peptides presented at the cell surface to CD8+ T cells. They can simply cut proteins or recombine their fragments thereby generating novel sequences, i.e. spliced peptides via a process called proteasome-catalysed peptide splicing (PCPS). In order to gain more insight into these processes, we here perform in vitro digestions of 55 synthetic polypeptide substrates by proteasome isoforms followed by LC-MS/MS measurements using different Mass Spectrometers.

2020-04-14 | PXD016782 | Pride

Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome

Project description:Spliced peptides are short protein fragments spliced together in the proteasome by peptide bond formation. True estimation of the contribution of proteasome-spliced peptides (PSPs) to the global Human Leukocyte Antigen (HLA) ligandome is critical. A recent study suggested that PSPs contribute up to 30% of the HLA ligandome. We performed a thorough reanalysis of the reported results using multiple computational tools and various validation steps and concluded that only a fraction of the proposed PSPs passes the quality filters. To better estimate the actual number of PSPs, we present an alternative workflow. We performed de-novo sequencing of the HLA-peptide spectra and discarded all de-novo sequences found in the UniProt database. We checked whether the remaining de-novo sequences could match spliced peptides from human proteins. The spliced sequences were appended to the UniProt fasta file, which was searched by two search tools at a FDR of 1%. We find that 2-6% of the HLA ligandome could be explained as spliced protein fragments. The majority of these potential PSPs have good peptide-spectrum match properties and are predicted to bind the respective HLA molecules. However, it remains to be shown how many of these potential PSPs actually originate from proteasomal splicing events.

2018-09-03 | PXD010793 | Pride

Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome

2020-11-13 | MSV000086452 | MassIVE

Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8+ T cells in HIV-1 infection

Project description:Peptides generated by proteasome-catalyzed splicing of non-contiguous amino acid sequences have been shown to constitute a source of non-templated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8+ T cells are key mediators of human immunodeficiency virus type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly-applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I-bound peptides on HIV-infected cells. We demonstrate that HIV-1-derived spliced peptides comprise a novel, but relatively minor, component of the HLA-I-bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8+ T cell responses relatively infrequently during infection, CD8+ T cells primed by partially-overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically under-utilized during natural infection.

2019-11-25 | PXD015489 | Pride

20S Proteasome Hyperactivation enhances translation, lipid metabolism, proteostasis and longevity: a Proteo-transcriptimic analysis

Project description:Our study elucidates the broad-spectrum effects of proteasome hyperactivation on the proteome, demonstrating its substantial influence on the proteostasis network. Through comprehensive analyses, we identified a significant reorganization of the transcriptome, enhanced mRNA processing, and increased translation activity. The study further reveals the hyperactivation's impact on intrinsically disordered proteins, lipid synthesis, and biogenesis, evidenced by major changes in lipid droplets and peroxisome proliferation. Notably, we uncovered protective phenotypes against oxidative stress, primarily mediated by Super Oxide Dismutases (SODs), indicating an elevated stress response readiness. Additionally, our research highlighted the hyperactive proteasome's selective targeting of vitellogenins, crucial for lipid metabolism and implicated in the aging process of C. elegans. Enhanced ERAD-dependent degradation was observed, facilitating the clearance of senescent VIT-2 protein aggregates and promoting healthier aging. The α3ΔN mutant significantly reduced the quantity of human alpha-1 antitrypsin (ATZ) aggregates and delayed their formation in a C. elegans model for ATZ disease, suggesting its potential as an ERAD enhancer. These findings propose proteasome hyperactivation as a promising strategy for treating aggregation-prone diseases, offering novel avenues for drug development and therapeutic interventions against neurodegeneration.

2024-05-01 | GSE259419 | GEO