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A cell-based platform for the investigation of immunoproteasome subunit β5i expression and biology of β5i-containing proteasomes

ABSTRACT: The degradation of intracellular proteins is a dynamic and tightly regulated process. Most of such substrates is hydrolyzed by proteasomes. Different forms of proteasomes are known. The role of immunoproteasomes (iPs) and intermediate proteasomes (intPs) is emerging. Here, using a CRISPR-Cas9 nickase technology a panel of four cell lines was obtained. Cells of histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma and hepatocarcinoma origin were modified to express proteasomes with mCherry-tagged β5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the chimeric gene expression in modified cells is under control of endogenous regulatory mechanisms and was increased following IFN-γ and/or TNF-α stimulation. Fluorescent proteasomes are catalytically active and were distributed within the nucleus and cytoplasm of the modified cells. RNAseq revealed marginal differences in gene expression profile between modified and initial cell lines, predominate metabolic pathways and a pattern of expressed receptors were determined for each cell line. Using obtained cell-lines it was shown that anti-cancer drugs ruxolitinib, vincristine and gefetinib stimulate the expression of β5i-containing proteasomes which might have an impact on the disease prognosis. Taken together, obtained cell lines represent convenient platform to study the immunoproteasome gene expression, localization of iPs and intPs, association of proteasomes with other proteins and many other aspects of proteasome biology under the influence of various drugs and conditions in real time and in living cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE183592 | GEO | 2021/09/14

REPOSITORIES: GEO

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Lipid-anchored Proteasomes Control Membrane Protein Homeostasis

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2023-05-16 | GSE232308 | GEO

AKIRIN2 controls the nuclear import of proteasomes in vertebrates

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Project description:The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPactivated protein kinase (AMPK)-regulated endosomal sorting complex required for transport (ESCRT)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low-glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low-glucose stress. Here, we uncover additional features of the selective microautophagy of proteasomes in budding yeast. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appears to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition.We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion.

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AKIRIN2 controls the nuclear import of proteasomes in vertebrates

Project description:AKIRIN2 controls the nuclear import of proteasomes in vertebrates

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A cell-based platform for the investigation of immunoproteasome subunit β5i expression and biology of β5i-containing proteasomes

Project description:A cell-based platform for the investigation of immunoproteasome subunit β5i expression and biology of β5i-containing proteasomes

| PRJNA761383 | ENA

Digestion of a variety of synthetic peptides by proteasomes

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2020-04-14 | PXD016782 | Pride

Digestion of TSN2 and TSN89 synthetic peptides by proteasomes

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2022-12-19 | PXD025995 | Pride

Proteomic Analysis of Affinity-Purified Proteasomes Identifies a Suite of Assembly Chaperones in Arabidopsis

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2019-09-25 | PXD014610 | Pride

The proteasome regulator PSME4 reduces the response to immunotherapy by abrogating tumor immunogenicity in NSCLC

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2023-02-26 | GSE201460 | GEO

Effect of LMP7 and MECL1-immunoproteasome subunits deficiency on the transcriptome of mouse bone marrow-derived dendritic cells

Project description:As regulators of protein degradation, proteasomes regulate practically all cellular functions. It is therefore logical to assume that replacement of the constitutive proteasome (CP) by its IFN- inducible homolog immunoproteasome (IP) could have far reaching effects on cell function. Accordingly, recent studies have revealed important roles for IPs in immune cells beyond MHC I-peptide processing. Moreover, the expression of IPs in non-immune cells from non-inflamed tissues suggests that the involvement of IPs is not limited to the immune system. We demonstrate here that IP-deficiency affects the transcription of 8104 genes in maturing dendritic cells (DCs). This occurs mainly through non-redundant regulation of key immune-related transcription factors by CPs and IPs. Additionally, IP-deficiency decreases DC's efficiency to activate CD8+ T cells in vivo. Our study reveals that the broad cellular roles of IPs could rely on transcription regulation and, more importantly, illustrates how IP-deficiency could generate MHC I-peptide processing-independent phenotypes.

2014-07-02 | GSE52616 | GEO

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