@article{53526f9c83f240c5bc2a6b7726b2ab4e,
title = "Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains",
abstract = "Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate 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 promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.",
keywords = "UBQLN2, liquid–liquid phase separation, polyphasic linkage, polyubiquitin, protein quality control",
author = "Dao, {Thuy P.} and Yiran Yang and Presti, {Maria F.} and Cosgrove, {Michael S.} and Hopkins, {Jesse B.} and Weikang Ma and Loh, {Stewart N.} and Casta{\~n}eda, {Carlos A.}",
note = "Funding Information: This work was supported by ALS Association grant 18‐IIP‐400, NIH R01GM136946 (all protein purifications, turbidity assays, microscopy, and NMR experiments) and NSF CAREER MCB 1750462 (SEC‐MALS‐SAXS experiments) to C.A.C. S.N.L. acknowledges support from NIH R01GM115762. M.S.C. acknowledges support from NIH R01CA140522. NMR data were acquired on an 800 MHz NMR spectrometer funded by NIH‐shared instrumentation grant 1S10OD012254. FRAP data were acquired at the Syracuse University Blatt BioImaging Center on a Zeiss LSM980 with Airyscan2 funded by NIH S10 OD026946‐01A1. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090‐01 from NIGMS. This study made use of NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR), which is supported by NIH grant P41GM111135 (NIGMS). We thank Chris Waudby for insightful conversations on binding affinities and Rohit Pappu, Kiersten Ruff, Tanja Mittag, Daniel Kraut, Elliot Dine, and Susan Krueger for stimulating discussions over the years leading to this project. We also thank Ashley Canning with assistance on AUC experiments. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health. Funding Information: This work was supported by ALS Association grant 18-IIP-400, NIH R01GM136946 (all protein purifications, turbidity assays, microscopy, and NMR experiments) and NSF CAREER MCB 1750462 (SEC-MALS-SAXS experiments) to C.A.C. S.N.L. acknowledges support from NIH R01GM115762. M.S.C. acknowledges support from NIH R01CA140522. NMR data were acquired on an 800 MHz NMR spectrometer funded by NIH-shared instrumentation grant 1S10OD012254. FRAP data were acquired at the Syracuse University Blatt BioImaging Center on a Zeiss LSM980 with Airyscan2 funded by NIH S10 OD026946-01A1. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090-01 from NIGMS. This study made use of NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR), which is supported by NIH grant P41GM111135 (NIGMS). We thank Chris Waudby for insightful conversations on binding affinities and Rohit Pappu, Kiersten Ruff, Tanja Mittag, Daniel Kraut, Elliot Dine, and Susan Krueger for stimulating discussions over the years leading to this project. We also thank Ashley Canning with assistance on AUC experiments. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.",
year = "2022",
month = aug,
day = "3",
doi = "10.15252/embr.202255056",
language = "English (US)",
volume = "23",
journal = "EMBO Reports",
issn = "1469-221X",
publisher = "Nature Publishing Group",
number = "8",
}