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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: PXD034874 | Pride | 2024-07-05

REPOSITORIES: Pride

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			Action	DRS
	190709_AB_test_DDA_SEC_n_13_14.raw	Raw
	190709_AB_test_DDA_SEC_n_25.raw	Raw
	190709_AB_test_DDA_SEC_n_33.raw	Raw
	190709_AB_test_DDA_SEC_n_45.raw	Raw
	190714_AB_DIA_SEC_fr13_14.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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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:As the major protein degradation machinery of eukaryotic cells, the 26S proteasome is generally thought to localize in the nucleus and cytosol. A portion of proteasomes are known to associate with various membrane structures of the cell, the mechanism and biological meaning of which have been elusive. Here we show that N-myristoylation of the proteasome subunit Rpt2 is an evolutionarily conserved determinant of proteasome-membrane interaction. Loss of this modification leads to embryonic lethality in mice, significant reduction of migration ability in MEFs and profound changes in the membrane-associated proteome as determined by SILAC-MS, suggesting a key role of membrane-tethered proteasomes in carrying out compartmentalized protein degradation. And the tumorigenicity is reduced in the oncogene-transformed MEF without modification. Serendipitously, we found that the Rpt2-G2A mutation cell lines confers partial resistance to proteasome inhibitors, such as Bortezomib and MG132. Thus, N-myristoylation of Rpt2 determines the localization and activity of the proteasome at the membrane, which is critical for embryogenesis, cellular homeostasis and tumorigenesis.

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Project description:AKIRIN2 regulates the import of proteasomes into the nucleus. To analyze AKIRIN2-containing proteasome complexes, we performed co-immunoprecipitation of GST-tagged AKIRIN2, followed by sucrose gradient centrifugation and mass spectrometry.

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