In-gel proteasome assay to determine the activity, amount, and composition of proteasome complexes from mammalian cells or tissues

Yazgili, Ayse Seda; Meul, Thomas; Welk, Vanessa; Kammerl, Ilona; Meiners, Silke

doi:10.1016/j.xpro.2021.100526

Cited by 28 publications

(30 citation statements)

References 9 publications

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“…1B). To study the assembly of the proteasome complex during ferroptosis, we performed native PAGE, during which the tertiary structure of the proteasome is preserved [20]. Confirming the findings of the SDS-PAGE, both 30S and 20S proteasomal activity and expression were lower after the induction of ferroptosis by erastin and RSL3 (Fig.…”

Section: Results: 31 Proteasome Function Is Inhibited During Ferroptosissupporting

confidence: 62%

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NFE2L1-mediated proteasome function protects from ferroptosis

Kotschi¹,

Jung²,

Willemsen³

et al. 2021

Preprint

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ObjectiveFerroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood.Methods and ResultsHere we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis.ConclusionOur data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.Graphical abstractHighlightsProteasome function is diminished during ferroptosisNFE2L1-mediated proteasomal activity protects from ferroptosisThe ubiquitination of the GPX4-glutathione pathway is implicated in Nfe2l1 deficiencyNFE2L1 deficiency in brown fat is associated with hallmarks of ferroptosis

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Section: Results: 31 Proteasome Function Is Inhibited During Ferroptosissupporting

confidence: 62%

“…The protocol for in-gel proteasome activity assay and subsequent immunoblotting was described previously [20]. In short, cells were lysed in OK Lysis buffer (50 mM Tris/HCl (pH 7.5), 2 mM DTT, 5 mM MgCl2, 10 % glycerol, 2 mM ATP, 0.05 % Digitonin), containing phosphate inhibitor (PhosStop, Roche).…”

Section: Native Page and Proteasomal Activitymentioning

confidence: 99%

NFE2L1-mediated proteasome function protects from ferroptosis

Kotschi¹,

Jung²,

Willemsen³

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…In line with the compromised proteasomal activity during the early stages of ferroptosis, the expression of key regulatory proteasomal subunits was lower with erastin and RSL3 treatment ( Figure 1 B). To study the assembly of the proteasome complex during ferroptosis, we performed native PAGE, during which the tertiary structure of the proteasome is preserved [ 20 ]. Confirming the findings of the SDS-PAGE, both 30S and 20S proteasomal activity and expression were lower after the induction of ferroptosis by erastin and RSL3 ( Figure 1 C), indicating acute impairment of proteasomal function during the induction of ferroptosis.…”

Section: Resultsmentioning

confidence: 99%

“…The protocol for in-gel proteasome activity assay and subsequent immunoblotting was described previously [ 20 ]. In short, cells were lysed in OK Lysis buffer (50 mM Tris/HCl (pH 7.5), 2 mM DTT, 5 mM MgCl 2 , 10% glycerol, 2 mM ATP, 0.05% Digitonin), containing phosphate inhibitor (PhosStop, Roche).…”

Section: Methodsmentioning

confidence: 99%

NFE2L1-mediated proteasome function protects from ferroptosis

Kotschi¹,

Jung²,

Willemsen³

et al. 2022

Molecular Metabolism

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Objective Ferroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. Methods and results Here we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4 -deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. Conclusion Our data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

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“…Native gel analysis and subsequent immunoblotting with an antibody detecting the 20S α1–7 subunits (ab22674; Abcam, Cambridge, UK) was performed as described previously [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

Activation of immune cell proteasomes in peripheral blood of smokers and COPD patients: implications for therapy

et al. 2021

Self Cite

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Immune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in COPD thereby affecting immune cell responses.We here analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMC) isolated from healthy male young smokers as well as from patients with severe COPD and compared them to matching controls. Proteasome expression was upregulated in COPD patients as assessed by RT-qPCR and mass spectrometry-based proteomics analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modeling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro. Inhibition of the immunoproteasome reduced proinflammatory cytokine expression in COPD-derived blood immune cells.Given the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD.

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In-gel proteasome assay to determine the activity, amount, and composition of proteasome complexes from mammalian cells or tissues

Cited by 28 publications

References 9 publications

NFE2L1-mediated proteasome function protects from ferroptosis

NFE2L1-mediated proteasome function protects from ferroptosis

NFE2L1-mediated proteasome function protects from ferroptosis

Activation of immune cell proteasomes in peripheral blood of smokers and COPD patients: implications for therapy

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