Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

Menneteau, Thomas; Fabre, Bertrand; Garrigues, Luc; Stella, Alexandre; Živković, Dušan; Roux‐Dalvai, Florence; Mouton-Barbosa, Emmanuelle; Beau, Mathilde; Renoud, Marie-Laure; Amalríc, François; Sensebé, Luc; Peredo, Anne Gonzalez de; Ader, Isabelle; Burlet‐Schiltz, Odile; Bousquet, Marie‐Pierre

doi:10.1074/mcp.ra118.000958

“…Similarly, we could not detect in yeast the chromosomally tagged FLAG-His6-PSMB2 subunit 74 . Lastly, in both rabbit and rat 20S proteasome complexes that were purified from liver tissue, immunoproteasome subunits were also identified, in line with the high abundance of immunoproteasome in liver samples 75 .…”

Section: Identification Of a New Psma7 Proteoformsupporting

confidence: 52%

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Vimer¹,

Nissan²,

Morgenstern³

et al. 2020

Preprint

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Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

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“…However, this subunit was not detected at all for the yeast complex. Similarly, we could not detect in yeast the chromosomally tagged FLAG-His6-PSMB2 subunit 74 . Lastly, in both rabbit and rat 20S proteasome complexes that were purified from liver tissue, immunoproteasome subunits were also identified, in line with the high abundance of immunoproteasome in liver samples 75 .…”

Section: Identification Of a New Psma7 Proteoformsupporting

confidence: 52%

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Vimer¹,

Nissan²,

Morgenstern³

et al. 2020

Preprint

View full text Add to dashboard Cite

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.

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“…The presence of int-proteasomes as normal constituents in different tissues has been established before as they have been reported to constitute up to 50 % of the total proteasome pool, depending on the tissue (16, 24), but a comprehensive investigation of proteasome composition in primary β cells or β cell models in non-stimulated conditions has not been performed. The Human Protein Atlas RNA-seq data indicated constitutive expression of the inducible β1i, β2i and β5i subunits in human pancreas, with 10 % of the RNA sequencing reads originating from the islets of Langerhans and 75 % coming from exocrine glandular cells (46).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, induction of expression of such proteasomes upon exposure of human pancreatic islets and rat and mouse insulinoma cells to INFγ and β but not toxic concentrations of IL-1β, was reported (18, 20). Furthermore, int-proteasomes (but not i-proteasomes) are constitutively expressed in various cells, including liver, heart, kidney, lung or colon (16, 21–24). They constitute between 1 % (heart) to 50 % (liver) of the total proteasome pool (16, 21, 23, 24).…”

Section: Introductionmentioning

confidence: 99%

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The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, non-toxic concentrations of interleukin 1 β

Khilji

¹

,

Verstappen

²

,

Dahlby

³

et al. 2019

Preprint

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Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

Cited by 21 publications

References 79 publications

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, non-toxic concentrations of interleukin 1 β

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