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Role for ribosomal quality control in sampling proteins for MHC class I-mediated antigen presentation

ABSTRACT: Mammalian cells present a fingerprint of their proteome to the adaptive immune system through the display of endogenous peptides on MHC-I complexes. MHC-I peptides originate from protein degradation by the proteasome, suggesting that efficiently-folding, long-lived proteins could evade monitoring. Here, we investigate the role of the ribosomal quality control (RQC) pathway, responsible for the degradation of nascent chains stalled at the ribosome during translation, in sampling proteins for antigen presentation. We find that degradation and presentation on MHC-I of model proteins encoded by defective mRNAs is independent of their folding potential. Quantitative profiling of MHC-I peptides in wild-type and RQC-deficient cells by mass spectrometry showed that RQC substantially contributes to the composition of the immunopeptidome. Our results not only reveal the role of RQC in sampling proteins that are otherwise difficult to present due to their folding properties, but also identify endogenous substrates of the RQC pathway in human cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Mario Oroshi

LAB HEAD: F. Ulrich Hartl

PROVIDER: PXD014644 | Pride | 2020-01-29

REPOSITORIES: Pride

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			Action	DRS
	20180228_QX0_MaPe_SA_P509_HeLa_KO_1.raw	Raw
	20180228_QX0_MaPe_SA_P509_HeLa_KO_2.raw	Raw
	20180228_QX0_MaPe_SA_P509_HeLa_KO_3.raw	Raw
	20180228_QX0_MaPe_SA_P509_HeLa_KO_4.raw	Raw
	20180228_QX0_MaPe_SA_P509_HeLa_KO_5.raw	Raw

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Project description:All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) can convey non-self antigens derived from pathogens or mutations to amount T-cell responses. Alterations in tumor-specific antigens – particularly mutation-bearing peptides (neoantigens) presented by MHC — can serve as potent substrates for anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring interferon gamma (IFN-γ) induced alterations to antigen presentation, using a lymphoma cell line. IFN-γ treatment resulted in a set of differentially expressed proteins (2 % of all quantified proteins) including components of antigen presentation machinery or interferon signaling pathways. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells antigen repertoires. Accordingly, by surveying immunopeptides, distinct peptide repertoires were exclusively observed in the IFN-γ induced samples. Furthermore, an additional set of presented peptides distinguished control and the IFN-γ samples by their altered relative abundances including neoantigens. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance should help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.

Project description:ABSTRACT Stimulating the commitment of implanted dystrophin+ muscle derived stem cells (MDSC) into myogenic, as opposed to lipofibrogenic, lineages is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSC from wild type (WT), myostatin knockout (Mst KO), and mdx (DMD model) (mdx) young mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSC to repair the injured gastrocnemius in aged mdx mice with exacerbated lipofibrosis. Surprisingly, the potent in vitro myotube formation by WT MDSC was refractory to modulators of myostatin expression or activity, and the Mst KO and mdx MDSC failed to form myotubes under any condition, despite all MDSC expressed Oct-4 and various stem cell genes and differentiated into other lineages. The genetic inactivation of myostatin or dystrophin in MDSC was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSC implanted into the injured gastrocnemius of old mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSC in vivo also significantly improved myofiber repair, but had no significant effects on lipofibrotic degeneration. In conclusion, while WT MDSC are considerably myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro but the myogenic capacity is recovered in vivo under the influence of the host tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSC. Key words: dystrophin, mdx mouse, Duchenne, fibrosis, dystrophy One sample each of mouse wild type (WT), myostatin knockout (KO), muscular dystrophy model mdx, and the transgenic OCT4 promoter plus reporter were grown, RNA was isolated, and subjected to the SABiosciences mouse stem cell oligo array.

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Role for ribosomal quality control in sampling proteins for MHC class I-mediated antigen presentation

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