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

Aleksandar Bartolome, Julia C. Heiby, Therese Dau, Domenico Di Fraia, Ivonne Heinze, Joanna M. Kirkpatrick, Alessandro Ori

Posted on: 1 May 2023

Preprint posted on 9 August 2022

ProteasomeID uses proximity labeling to identify the interaction between proteasome and membrane-associated protein or transcription factor, which are mostly undetectable and challenging under traditional approaches.

Selected by Aniruddha Das

Background:

The cellular ubiquitin-proteasome system (UPS) can be modulated to develop novel therapeutic modalities for the degradation of disease-associated proteins. Proteasome inhibitors have been used clinically, and the ongoing development of small molecule proteolysis targeting chimeras (PROTACs) indicates its valuable therapeutic opportunities. The development of an effective protein degrader is challenging because proximity induction between ubiquitin ligase and the target protein not always led to ubiquitylation followed by degradation. An analytic approach that would allow one to monitor the endogenous or chemical compound-assisted target protein association with the cellular proteasome, could drastically improve degrader development. Furthermore, heterogeneity due to the variable stoichiometry of subunits, and localization of proteasomes in the different cellular compartments indicate the broad array of proteasome interactomes. Hence, the development of a method capable of detecting such complex interactions is valuable in the field of targeted protein degradation. Previous study indicates that tissue type-specific expression of requisite ligase limits the targeted degradation via selective ubiquitin ligase. Targeted protein degradation through direct substrate recruitment to the proteasome could be a promising approach. In this preprint, ProteasomeID is introduced as a novel quantifiable approach that allows the detection of interacting proteins and substrates, and the ability of small molecule degraders that induce the recruitment of target proteins to proteasomes within cells.

 

What I like about the preprint and key findings:

The promiscuous biotin ligase BirA fusion at the C-terminal of the core particle protein PSMA4 or the regulatory particle protein PSMC2 allowed conjugating biotin to proteins that come into ~10 nm proximity of the bait protein on the proteasome structure. MiniTurbo was used over BirA in the mouse model for its relatively higher biotinylation efficiency. Biotin-conjugated proteins were further captured from the cellular or tissue lysate using a streptavidin enrichment protocol and analyzed by deep Data Independent Acquisition mass spectrometry. Using this approach, the authors showed that BirA fusion did not significantly alter the assembly and endogenous activity of the cellular Proteasome, which makes the tool useful to study UPS. Importantly, ProteasomeID could be beneficial in expanding the repertoire of proteasome interactors, as this approach allows the detection of stronger interactors like the COP signalosome complex and transient interactors like USP14, a reversibly associating subunit of the 19S particle. Moreover, ProteasomeID as a novel tool could help monitor the altered dynamics in proteasome interactions during proteotoxic stress or in the disease model, and also quantify proteasome interacting proteins in different tissues or organs of the mouse model.

Primarily, ProteasomeID allowed the authors to reveal novel interactors of the proteasome, like phosphatidylinositol phosphatase Synaptojanin 1 (SYNJ1) which is challenging to detect when using the conventional immunoprecipitation assay, also indicating an unanticipated role in vascular trafficking and phospholipid metabolism. Moreover, the authors successfully established a mouse model for ProteasomeID, which allowed them to quantify the proteasome interacting proteins in multiple tissues. The expression of the tagged proteasome subunit did not negatively impact the mouse. However, a relatively lower level of the tagged proteasome was found in the brain and heart. The authors speculated that this might be due to the differences in protein turnover among organs. Also, the author proposed that prolonged doxycycline (Dox) induction might enable higher biotin ligase incorporation into proteasomes in an organ, like the brain. Further, by using KB02-JQ1 PROTAC which targets bromodomain-containing proteins (BRDs),  the authors could show the prominent enrichment of BRD-containing proteins upon proteasome inhibition and biotin supplementation, indicating the specificity of ProteasomeID.

Figure 1.

Panel A from Figure 1 of Bartolome et al. (2022). Figure reproduced under a CC-BY-NC-ND 4.0 International License. A) Schematic representation of fused biotin ligase to proteasome subunit for ProteasomeID.

Panel A from Figure 5 of Bartolome et al. (2022). Figure reproduced under a CC-BY-NC-ND 4.0 International License. B) ProteasomeID mouse model. Dox-inducible mouse line generated by crossing lox-STOP-lox; PSMA4-miniTurbo with Cre recombinase under the control of ubiquitous CMV promoter line.

Panel A from Figure S2A of Bartolome et al. (2022). Figure reproduced under a CC-BY-NC-ND 4.0 International License. C) Schematic of optimized BioID protocol.

 

Future aspects of the study:

The authors of this preprint propose crossing mouse expressing the Cre recombinase under the control of specific drivers with a ProteasomeID mouse, which could allow the study of proteasome interactions in a tissue-specific manner. Secondly, a combination of proximity labeling and proteasome inhibition could help identify proteasome activators, endogenous substrates, and transcription factors that are challenging to quantify. Thirdly, ProteasomeID could be useful in monitoring the perturbation of cellular proteostasis by proteotoxic stress or to monitor altered dynamics in proteasome interactions and substrates. Moreover, ProteasomeID could help to directly identify the known targets of PROTACs, and further, assess the efficacy of protein degraders in vivo.

 

Questions and suggestions for the authors:

  1. As part of this study, SYNJ1 has been uncovered as a novel interactor indicating the role of the proteasome in vascular trafficking and phospholipid metabolism. It would be really interesting to study the enrichment of SYNJ1 upon proteasomal inhibition or in the presence of MG132, which would allow a further understanding of the fate of SYNJ1 upon proteasome interaction.
  2. The Proteasome can differ based on the composition of catalytic subunits, like constitutive, immunoproteasomes, intermediate proteasomes, thymoproteasomes, and Spermatoproteasomes. Have the authors considered this heterogeneity and tried fusing biotin ligase with other subunits without compromising the proteasome activity in ProteasomeID, which might result in a much broader applicability?
  3. Degrader molecules could recruit substrates directly to the proteasome with ubiquitination, allowing proteins to degrade via a universal degradation strategy without available lysines. Could the authors perhaps comment on the ubiquitination status of the interacting partners of the proteasome while using ProteasomeID, to indicate an alternative role of the proteasome?

 

References:

Bashore C, Prakash S, Johnson MC, Conrad RJ, Kekessie IA, Scales SJ, Ishisoko N, Kleinheinz T, Liu PS, Popovych N, Wecksler AT, Zhou L, Tam C, Zilberleyb I, Srinivasan R, Blake RA, Song A, Staben ST, Zhang Y, Arnott D, Fairbrother WJ, Foster SA, Wertz IE, Ciferri C, Dueber EC. Targeted degradation via direct 26S proteasome recruitment. Nat Chem Biol. 19, 55–63 (2023). https://www.nature.com/articles/s41589-022-01218-w

Chen X, Htet ZM, López-Alfonzo E, Martin A, Walters KJ. Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies. FEBS J. 288: 5231–5251 (2021). https://doi.org/10.1111/febs.15638

Erales J, Coffino P. Ubiquitin-independent proteasomal degradation. Biochim Biophys Acta. 1843:216–21 (2014). https://doi.org/10.1016/j.bbamcr.2013.05.008

Garber K. The PROTAC gold rush. Nat Biotechnol. 40, 12–16 (2022). https://www.nature.com/articles/s41587-021-01173-2

Mackmull MT, Klaus B, Heinze I, Chokkalingam M, Beyer A, Russell RB, Ori A, Beck M. Landscape of nuclear transport receptor cargo specificity. Mol Syst Biol. 13:962 (2017). https://doi.org/10.15252/msb.20177608

Morozov AV, Karpov VL. Biological consequences of structural and functional proteasome diversity. Heliyon. 4:e00894 (2018). https://doi.org/10.1016/j.heliyon.2018.e00894

Osei-Amponsa V, Walters KJ. Proteasome substrate receptors and their therapeutic potential. Trends Biochem Sci. 47, 950–964 (2022). https://doi.org/10.1016/j.tibs.2022.06.006

Rousseau A, Bertolotti A. Regulation of proteasome assembly and activity in health and disease. Nat Rev Mol Cell Biol. 19, 697–712 (2018). https://www.nature.com/articles/s41580-018-0040-z

Tags: proteasome, protein degradation, proximity labeling

doi: https://doi.org/10.1242/prelights.34428

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