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Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

Thuy P. Dao, Yiran Yang, Michael S. Cosgrove, Jesse B. Hopkins, Weikang Ma, View ORCID ProfileCarlos A. Castañeda
doi: https://doi.org/10.1101/2021.11.12.467822
Thuy P. Dao
1Departments of Biology and Chemistry, Syracuse University, Syracuse, NY 13244, USA
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Yiran Yang
2Department of Chemistry, Syracuse University, Syracuse University, Syracuse, NY 13244, USA
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Michael S. Cosgrove
3Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
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Jesse B. Hopkins
4The Biophysics Collaborative Access Team (BioCAT), Department of Biological Sciences, Illinois Institute of Technology, Chicago, IL 60439, USA
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Weikang Ma
4The Biophysics Collaborative Access Team (BioCAT), Department of Biological Sciences, Illinois Institute of Technology, Chicago, IL 60439, USA
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Carlos A. Castañeda
1Departments of Biology and Chemistry, Syracuse University, Syracuse, NY 13244, USA
5Interdisciplinary Neuroscience Program, Syracuse University, Syracuse University, Syracuse, NY 13244, USA
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  • ORCID record for Carlos A. Castañeda
  • For correspondence: cacastan@syr.edu
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Summary

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. However, interactions with ubiquitin or multivalent K48-linked chains eliminate LLPS. Here, we found that, although some polyubiquitin chain types (K11-Ub4 and K48-Ub4) did generally inhibit UBQLN2 LLPS, others (K63-Ub4, M1-Ub4 and a designed tetrameric ubiquitin construct) significantly enhanced LLPS. Using nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in chain conformations, but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin binding surface significantly promoted UBQLN2 LLPS by enabling a switch between homotypically to partially heterotypically-driven phase separation. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic phase separation with ubiquitin-binding shuttles and adaptors.

  • Ubiquitin or short polyubiquitin chains bind to phase separation-driving stickers on UBQLN2 and inhibit its phase separation whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation.

  • Compact K11- and K48-linked Ub4 chains destabilized UBQLN2 phase separation, while extended M1- and K63-linked Ub4 promoted UBQLN2 phase separation.

  • Chain conformation and accessibility of the Ub interacting surface is a driving factor of UBQLN2/polyUb co-phase separation.

  • UBQLN2 condensates assemble during in vitro enzymatic assembly of K63-linked polyUb chains as free ubiquitin is depleted.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 13, 2021.
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Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages
Thuy P. Dao, Yiran Yang, Michael S. Cosgrove, Jesse B. Hopkins, Weikang Ma, Carlos A. Castañeda
bioRxiv 2021.11.12.467822; doi: https://doi.org/10.1101/2021.11.12.467822
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Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages
Thuy P. Dao, Yiran Yang, Michael S. Cosgrove, Jesse B. Hopkins, Weikang Ma, Carlos A. Castañeda
bioRxiv 2021.11.12.467822; doi: https://doi.org/10.1101/2021.11.12.467822

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