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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

ABSTRACT: Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell

SUBMITTER: Emilio Cirri

LAB HEAD: Alessandro Ori

PROVIDER: PXD033008 | Pride | 2024-07-05

REPOSITORIES: Pride

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			Action	DRS
	20220104_103138_261221_AB_mTurboID_MG132_exp_AmBic_dDIA.sne	Other
	20220104_103506_261221_AB_mTurboID_MG132_exp_AmBic_dDIA_Report.xls	Xls
	20220404_161929_261221_AB_mTurboID_exp_AmBic_dDIA_Peptide_Report.xls	Xls
	211221_AB_BioID_Ambic_A4_mT_MG_10.raw	Raw
	211221_AB_BioID_Ambic_A4_mT_MG_13.raw	Raw

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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Project description:Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.

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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

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Chronic administration of non-constitutive proteasome inhibitor modulates plasticity in hippocampus following tetanic stimulation, but not short theta-burst stimulation.

Project description:Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Proteasome inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here we studied the effect of chronic administration of non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. The TBS stimulation did not change LTP kinetics in hippocampal slices, however chronic administration of ONX-0914 led to the decrease in fEPSP slopes after tetanic stimulation. Observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP but not the LTP occurring after TBS, supporting the relevance and complexity of the role of proteasomes in the synaptic plasticity.

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Project description:Disruption in proteostasis is a hallmark of cancer, with aberrations in proteasome-mediated degradation highly implicated in this malignancy. Proteasomes further bear critical importance in tumor immunogenicity by regulating inflammatory signaling, promoting immune cell activation and playing a crucial role in antigen processing and presentation. Indeed, response to immunotherapy in melanoma patients has been recently linked to increased expression of immunoproteasomes, a specialized form of proteasomes that are upregulated under inflammatory conditions. Yet, the percentage of patients that respond to therapy remains low, and the underlying mechanisms driving resistance remain poorly understood. Further, comprehensive analysis of the role proteasomes play in the pathophysiology of cancer and as a modulator of anti-tumor immunity is remains lacking. Here, we show that proteasome complex composition varies across cancer types and can be used to stratify patient response to immunotherapy. Specifically, we found that the proteasome regulator PSME4 is upregulated in NSCLC and associated with poor prognosis. We show that PSME4 alters proteasome activity and antigen processing attenuating the antigenic diversity and presentation. Through exacting biochemical assays, proteasome profiling of patient-derived NSCLC samples, combined with scRNA-seq, immunopeptidomics and in vivo analyses we uncovered a novel mechanism of immune evasion mediated by PSME4, which promotes an immunosuppressive tumor microenvironment and abrogates anti-tumor immunity. Collectively, our approach may be adapted to other cancer types and pathological settings and affords a novel paradigm by which proteasome composition heterogeneity should be examined and targeted in the context of precision oncology.

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Project description:Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Pharmacological proteasomal inhibitors such as lactacystin can induce apoptosis in cultured mouse cortical neurons through the activation of caspase-3. However, Meiners et al., 2003 suggested that low dose of lactacystin induces a concerted expression of proteasome genes and de novo formation of proteasomes.

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TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells

Project description:Yes-associated protein (YAP), the downstream transducer of the Hippo pathway is a key regulator of organ size, differentiation and tumourigenesis. Yet, the activity of YAP can also be modulated by Hippo-independent functions. To disclose these Hippo-independent regulators, we performed a genome-wide CRISPR screening approach that identified the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. Using RNA-Sequencing and ChIP-Sequencing approaches we show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. Mechanistically, TRPS1 represses YAP-dependent enhancers function by recruiting a spectrum of corepressor complexes to joint sites. Consequently, loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer which is associated decreased YAP activity and leads to a decreased frequency of intratumoural immune cells. Consistently, depletion of TRPS1 in the syngeneic 4T1 tumour model leads to a strongly decreased tumour growth in vivo. Our study uncovers TRPS1 as a new epigenetic regulator of YAP activity and it connects repression of YAP-dependent enhancer function to breast cancer. For project-related queries, please contact bjoern.voneyss@leibniz-fli.de <mailto:bjoern.voneyss@leibniz-fli.de>.

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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.

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