ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies
Posted on: 1 May 2023
Preprint posted on 9 August 2022
Article now published in eLife at http://dx.doi.org/10.7554/elife.93256.3
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 DasCategories: biochemistry, cell biology, molecular biology
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:
- 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.
- 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?
- 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
doi: https://doi.org/10.1242/prelights.34428
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the biochemistry category:
Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia
Gustavo Stelzer, Marcus Oliveira
Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice
Gustavo Stelzer, Marcus Oliveira
Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping
Bhaval Parmar
Also in the cell biology category:
Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder
Harvey Roweth
Investigating Mechanically Activated Currents from Trigeminal Neurons of Non-Human Primates
Vanessa Ehlers
Motor Clustering Enhances Kinesin-driven Vesicle Transport
Sharvari Pitke
Also in the molecular biology category:
Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder
Harvey Roweth
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Jonathan Townson
preListsbiochemistry category:
in theBSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
Peer Review in Biomedical Sciences
Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biomedical sciences deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.
List by | Marcus Oliveira et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
Also in the cell biology category:
November in preprints – the CellBio edition
This is the first community-driven preList! A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. Categories include: 1) cancer cell biology 2) cell cycle and division 3) cell migration and cytoskeleton 4) cell organelles and organisation 5) cell signalling and mechanosensing 6) genetics/gene expression
List by | Felipe Del Valle Batalla et al. |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |