An official website of the United States government
Abstract Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N‐terminal ubiquitin‐like (UBL) and C‐terminal ubiquitin‐associated (UBA) domains, respectively. Between these two folded domains are low‐complexity STI1‐I and STI1‐II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1‐II region enables UBQLN2 to undergo liquid–liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well‐studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1‐I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.
more »
« less
Ubiquitin‐Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?
Abstract Liquid‐liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin‐binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.
more »
« less
- Award ID(s):
- 1750462
- PAR ID:
- 10455541
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- BioEssays
- Volume:
- 42
- Issue:
- 11
- ISSN:
- 0265-9247
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2’s phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.more » « less
-
Liquid–liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.more » « less
-
Abstract Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes.more » « less
-
Some proteins, including yeast translation termination factor Sup35 (eRF3) are capable of both stress-induced liquid-liquid phase separation (LLPS) and formation of solid fibrous aggregates (amyloids). Fragmentation and propagation of amyloid fibrils generates transmissible (in yeast, heritable) self-perpetuating protein agents, termed prions. Relationships between these processes are still poorly understood. Previous literature data suggested that the ability of Sup35 orthologs to form a prion is sporadically distributed in fungal evolution, and depends on amino acid composition of Sup35 prion domain (PrD), rather than on a evolutionarily variable specific sequence. We have studied two groups of proteins: 1) fungal Sup35 PrDs of various evolutionary origins, and 2) artificially synthesized “scrambled” variants of Saccharomyces cerevisiae Sup35 PrD, that possess identical amino acid composition but different sequences. These proteins were fused to fluorophores and expressed in S. cerevisiae cells. LLPS and amyloid/prion formation were assessed by fluorescence microscopy and biochemical approaches. Amino acid sequences were analyzed by various computational algorithms. Our data indicates that propagation of prion state strongly depends on the evolutionary distance from the host. In contrast, majority of proteins studied are capable of both LLPS and ability to form amyloid fibrils. These capabilities are associated with specific patterns of PrD amino acid distribution, that are broadly conserved among fungi. Notably, PrDs of different sequences differ from each other by their ability to convert from liquid condensates to amyloids, and relationship between these processes is apparently optimized in evolution. Moreover, heterotypic PrDs are can colocalize with each other within liquid condensates and influence amyloid conversion by each other. To conclude, LLPS and amyloid properties depend on specific evolutionarily conserved sequence patterns, indicating possible important biological roles for these processes. These patterns could potentially be used to predict LLPS and prion potential in other sequence contexts. This work was supported by NSF grant 2345660.more » « less
