Lipid-anchored Proteasomes Control Membrane Protein Homeostasis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE232308 | GEO | 2023/05/16
REPOSITORIES: GEO
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Project description:The 26S proteasome is an essential protease for the selective elimination of dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this multi-catalytic, ATP-dependent proteolytic machine in plants, we exploited Arabidopsis particles tagged at either the core protease (CP) or regulatory particle (RP) sub-complexes for rapid affinity purification followed by deep sequence analysis via mass spectrometry. Studies on proteasomes purified from whole seedlings via either a CP or RP subunit with or without ATP, which is needed to maintain the holo-proteasome complex, identified all known core subunits but failed to detect subunit isoform preferences, suggesting that Arabidopsis does not construct unique proteasomes sub-types. We also identified a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Analysis of proteasomes enriched from seedlings exposed to the proteasome inhibitor MG132 revealed the accumulation of novel assembly intermediates, which reflect partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Unlike in yeast, genetic analyses of Arabidopsis UMP1 showed that this chaperone is essential, with mutants missing the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single mutants impacting ump1a also accumulate a collection of proteasome assembly intermediates, consistent with its importance for CP construction. With this list of chaperones, it should now be possible to study the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more core subunits.
2019-09-25 | PXD014610 | Pride
Project description:The 26S proteasome regulates degradation of many cellular proteins to maintain cellular homeostasis. Disruption of proteasome activity followed by dysregulation of tumor suppressors and oncogenes is rampant in many cancers, hence chemical inhibitors of the proteasome have utility as cancer therapeutics, although the underlying mechanisms of their effects in the clinic is poorly understood. We have employed whole genome microarray expression profiling as a discovery platform to identify genes and potential signaling pathways that are affected MCF7 breast cancer cells are treated with MG132 a chemical inhibitor of the proteasome. Total RNA was collected from MCF7 breast cancer cells treated with DMSO (vehicle control) and 1 uM MG132 for 4 or 24H . We identified time dependent changes in the expression of genes enriched in p53 and estrogen receptor pathways, two signatures relevant to breast cancer biology.
2020-01-07 | GSE141858 | GEO
Project description: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.
2021-09-14 | GSE183592 | GEO
Project description:Lysosomes are a major site of intracellular acidic hydrolase-mediated proteolysis and cellular degradation in a membrane-enclosed organelle. Alternatively, soluble, ubiquitin-bound proteins are degraded via the proteasome, a megadalton protein complex within the cytoplasm. The interplay between both degradation pathways has been of increasing interest in recent years. To determine the effects of proteasome inhibition on the lysosomal compartment, we investigated enriched lysosomal fractions, autophagosomal fractions and immunoprecipitated proteasomes from HEK293 cells after proteasome inibition by MG132 or bortezomib in comparison to the respective fractions from control cells.
2020-04-20 | PXD015243 | Pride
Project description:Bruton’s tyrosine kinase (BTK) inhibitor Ibrutinib has been shown to synergize in vitro with proteasome inhibitors (PIs) in reducing the viability of cells derived from B-cell malignancies, but the mechanism is not known. We report here that an off-target effect of Ibrutinib causes synergy because not all BTK inhibitors exhibited the synergistic effect, and those that synergized did so even in cells that do not express BTK. The allosteric BTK inhibitor CGI-1746 showed the strongest synergy. Co-treatment of cells with CGI-1746 increased PI-induced expression of heat shock proteins and accumulation of ubiquitin conjugates. The CGI-1746 inhibited the degradation of a model substrate by a purified 26S proteasome. CGI-1746, but not other BTK inhibitors, allosterically inhibited ATPase activity and 2 and 1 peptidase activities of the 26S proteasomes. Although the effect is unlikely to contribute to the anti-neoplastic activity of BTK inhibitors in multiple myeloma and mantle cell lymphoma, it demonstrates a conceptually novel mode of inhibition that may aid the development of more potent proteasome inhibitors and improve response in solid tumors clinically.
2023-08-01 | GSE239482 | GEO
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.
2022-02-06 | PXD031469 |
Project description:The proteasome is the main proteolytic system for targeted protein degradation in the cell. Its function is fine-tuned according to cellular needs. Inhibition of the respiratory chain impairs proteasome activity, regulation of proteasome function by mitochondrial metabolism, however, is unknown. Here, we demonstrate that mitochondrial dysfunction reduces the assembly and activity of the 26S proteasome. Defects in respiratory chain caused metabolic reprogramming of the Krebs cycle and deficiency in the amino acid aspartate resulting in reduced 26S proteasome function. Aspartate supplementation fully restored assembly and activity of 26S proteasome complexes. This metabolic reprogramming involved sensing of aspartate via the mTORC1 pathway and the mTORC1-dependent transcriptional activation of defined proteasome assembly factors. Metabolic regulation of 26S function was confirmed in patient-derived skin fibroblasts with respiratory dysfunction containing a single mitochondrial mutation. Importantly, treatment of primary human lung fibroblasts with the respiratory chain inhibitor and anti-diabetic drug metformin similarly reduced assembly and activity of 26S proteasome complexes, which was fully reversible and rescued by supplementation of aspartate or pyruvate. Of note, reduced 26S activity conferred increased resistance towards the proteasome inhibitor Bortezomib, which was reversible upon pyruvate supplementation. Our study uncovers a fundamental novel mechanism of how mitochondrial metabolism adaptively adjusts protein degradation by the proteasome. It thus unravels unexpected consequences of defective mitochondrial metabolism in disease or drug-targeted mitochondrial reprogramming for proteasomal protein degradation in the cell. As metabolic inhibition of proteasome function can be alleviated by treatment with aspartate or pyruvate, our results also have therapeutic implications.
2020-07-26 | GSE153983 | GEO
Project description:Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
2021-08-27 | GSE157663 | GEO
Project description:Proteasomes are a critical component of quality control that regulates turnover of short-lived, unfolded, and misfolded proteins. Proteasome activity has been therapeutically targeted and considered as a treatment option for several chronic lung disorders including pulmonary fibrosis. Though pharmacologic inhibition of proteasome activity effectively prevents the transformation of fibroblasts to myofibroblasts, the effect on alveolar type 2 (AT2) epithelial cells is not clear. To address this knowledge gap, we generated a genetic model in which a proteasome subunit, RPT3, which promotes assembly of active 26S proteasome, was conditionally deleted in AT2 cells of mice. Targeted deletion of RPT3 in AT2 cells resulted in 26S proteasome dysfunction, leading to augmented cell stress and death. Acute loss of AT2 cells resulted in depletion of alveolar surfactant, disruption of the alveolar epithelial barrier and, ultimately, lethal acute respiratory distress syndrome. This study underscores the need for further investigation into the differential effect of proteasome inhibition in lung cell types.
2019-08-29 | GSE123873 | GEO
Project description:For many years, the ubiquitin-26S proteasome degradation pathway was considered the principal route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by an ubiquitin-independent mechanism mediated by the core 20S proteasome itself. The fact that half of cellular proteasomes are free 20S complexes suggests that degradation by this complex is not limited to rare cases. Identifying 20S proteasome substrates is challenging, as different pools of the same protein can be sent to degradation via either 20S or 26S proteasomes. Hence, current knowledge regarding the repertoire of 20S proteasome substrates mainly originates from individual case studies. Here, in an effort to unravel the global repertoire of substrates degraded by the 20S proteasome, we used an advanced mass spectrometry approach coupled with biochemical and cellular analysis. Our analysis enabled the identification of hundreds of 20S proteasome substrates. In addition to proteins that are degraded to completion, we also identified proteins that undergo specific cleavage by the 20S proteasome, at their N- or C- termini, to possibly tune their function. We also found that 20S substrates are significantly enriched with RNA- and DNA-binding proteins that contain intrinsically disordered regions. The vast majority of them are localized in the nucleus and stress granules. Further, we demonstrate that oxidized proteasomes have reduced proteolytic activity compared to naïve proteasomes, which we propose is an adaptive advantage under conditions of cellular stress. Whereas oxidized protein substrates, rather than being folded proteins that lost their native structure due to the stress, actually display a higher degree of structural-disorder than naïve proteins. In summary, here we shed light on the nature of the 20S substrates, providing critical insight into the biological role of the 20S proteasome.
2023-12-21 | PXD044399 | Pride