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

Dao, Thuy P.; Yang, Yiran; Presti, Maria F; Cosgrove, Michael S.; Hopkins, Jesse B.; Ma, Weikang; Loh, Stewart N.; Castañeda, Carlos

doi:10.15252/embr.202255056

Cited by 31 publications

(57 citation statements)

References 94 publications

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“…This is because the microscopic interactions and mesh structure of the dense phase are both much smaller than the molecules. In agreement with this expectation, the concentration of UBQLN2 molecules in the dense phase is nearly unchanged as polyUb is added (Dao et al, 2022).…”

Section: Theory Development For How Ub Ligands Inhibit and Promote Ph...supporting

confidence: 75%

Decoding optimal ligand design for multicomponent condensates

Galagedera

Dao

Enos

et al. 2023

Preprint

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Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub's ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

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Section: Theory Development For How Ub Ligands Inhibit and Promote Ph...supporting

confidence: 75%

Decoding optimal ligand design for multicomponent condensates

Galagedera

Dao

Enos

et al. 2023

Preprint

Self Cite

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show abstract

“…A recent study on the effects of ubiquitin linkage type on UBQLN2 LLPS indicated that while K48-linked chains slightly promote UBQLN2 LLPS at very low Ub:UBQLN2 concentrations, most concentrations of K48-linked Ub decreased the propensity for UBQLN2 to phase separate. In contrast, K63-linked Ub had a much broader range of concentrations that promoted LLPS (Dao et al ., 2022). As E6AP is known to catalyze K48-linked ubiquitin chains, we would expect ubiquitination of a substrate by E6AP to cause its exclusion from UBQLN2 LLPS droplets.…”

Section: Discussionmentioning

confidence: 99%

“…Of note, a recent publication found ubiquitin chain binding to the UBQLN2 UBA induces small shifts in the NMR signals of amino acids in the region of UBQLN2 homologous to UBAA, with the magnitude of shifting dependent on chain linkage type (i.e. M1, K11, K48, K63) (Dao et al ., 2022); this finding suggests different steric hindrances from the chain type impact the amount of UBAA reorganization. Thus, either ubiquitin or E6AP AZUL binding to UBA appears to be sensed at the UBAA domain.…”

Section: Discussionmentioning

confidence: 99%

“…The UBA in UBQLN2 contributes to UBQLN2 liquid-liquid phase separation (LLPS) (Dao et al , 2018; Zheng et al , 2021) and RAD23B’s two UBA domains similarly drive formation of phase-separated nuclear foci containing proteasomes (Yasuda et al ., 2020). K48-linked ubiquitin chains appear to drive formation of RAD23B liquid droplets (Yasuda et al ., 2020) and K48- or K63-linked ubiquitin chains slightly or strongly promote UBQLN2 LLPS, respectively (Dao et al , 2022).…”

Section: Introductionmentioning

confidence: 99%

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Discovery of E6AP AZUL binding to UBQLN1/2 in cells, phase-separated droplets, and an AlphaFold-NMR integrated structure

Buel

Chen

Myint

et al. 2022

Preprint

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The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 phase-separated droplets and E6AP interacts with UBQLN1/2 in cells. Steady-state and transfer NOE experiments indicate direct interaction of AZUL with the UBQLN1 UBA domain. Intermolecular contacts identified by NOESY data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We also identify a concentration-dependent oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA-adjacent, UBAA) that is alpha-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in droplets and cells.

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“…Importantly, ALS-associated mutations in UBQLN2 increase oligomerization, and the ubiquitin-binding generally disrupts LLPS, droplets, and aggregates ( Dao et al, 2019 ; Zheng et al, 2021 ). The effects of polyubiquitin chains on UBQLN2 LLPS are highly dependent on the linkage-types; thus, K11- and K48-ubiquitin chains inhibit LLPS, whereas K63- and M1-ubiquitin chains, which are extended and flexible, significantly enhance UBQLN2 LLPS ( Dao et al, 2022 ). In addition to UBQLN2, K63- and M1-chains also stabilize the LLPS of p62, an UBA-containing protein ( Sun et al, 2018 ; Zaffagnini et al, 2018 ).…”

Section: Heterologous Ubiquitination and Llpsmentioning

confidence: 99%

Involvement of heterologous ubiquitination including linear ubiquitination in Alzheimer’s disease and amyotrophic lateral sclerosis

Sato

Oikawa²,

Shimizu³

et al. 2023

Front. Mol. Biosci.

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In neurodegenerative diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), the progressive accumulation of ubiquitin-positive cytoplasmic inclusions leads to proteinopathy and neurodegeneration. Along with the seven types of Lys-linked ubiquitin chains, the linear ubiquitin chain assembly complex (LUBAC)-mediated Met1-linked linear ubiquitin chain, which activates the canonical NF-κB pathway, is also involved in cytoplasmic inclusions of tau in AD and TAR DNA-binding protein 43 in ALS. Post-translational modifications, including heterologous ubiquitination, affect proteasomal and autophagic degradation, inflammatory responses, and neurodegeneration. Single nucleotide polymorphisms (SNPs) in SHARPIN and RBCK1 (which encodes HOIL-1L), components of LUBAC, were recently identified as genetic risk factors of AD. A structural biological simulation suggested that most of the SHARPIN SNPs that cause an amino acid replacement affect the structure and function of SHARPIN. Thus, the aberrant LUBAC activity is related to AD. Protein ubiquitination and ubiquitin-binding proteins, such as ubiquilin 2 and NEMO, facilitate liquid-liquid phase separation (LLPS), and linear ubiquitination seems to promote efficient LLPS. Therefore, the development of therapeutic approaches that target ubiquitination, such as proteolysis-targeting chimeras (PROTACs) and inhibitors of ubiquitin ligases, including LUBAC, is expected to be an additional effective strategy to treat neurodegenerative diseases.

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

Cited by 31 publications

References 94 publications

Decoding optimal ligand design for multicomponent condensates

Decoding optimal ligand design for multicomponent condensates

Discovery of E6AP AZUL binding to UBQLN1/2 in cells, phase-separated droplets, and an AlphaFold-NMR integrated structure

Involvement of heterologous ubiquitination including linear ubiquitination in Alzheimer’s disease and amyotrophic lateral sclerosis

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