The pros and cons of ubiquitination on the formation of protein condensates

Abstract

Ubiquitination, a post-translational modification that attaches one or more ubiquitin (Ub) molecules to another protein, plays a crucial role in the phase-separation processes. Ubiquitination can modulate the formation of membrane-less organelles in two ways. First, a scaffold protein drives phase separation, and Ub is recruited to the condensates. Second, Ub actively phase-separates through the interactions with other proteins. Thus, the role of ubiquitination and the resulting polyUb chains ranges from bystanders to active participants in phase separation. Moreover, long polyUb chains may be the primary driving force for phase separation. We further discuss that the different roles can be determined by the lengths and linkages of polyUb chains which provide preorganized and multivalent binding platforms for other client proteins. Together, ubiquitination adds a new layer of regulation for the flow of material and information upon cellular compartmentalization of proteins.

Keywords: phase separation; polyubiquitin chain; post-translational modification; stress granule; ubiquitination.

Figure 1

Ub exists as covalently linked polymers (A) Ub is shown as cartoon, on which eight residues could be covalently linked to another Ub are shown in sticks (green) (left); The surface of Ub is shown with hydrophobic patches colored: F4 patch (light blue), I36 patch (green) and I44 patch (red); (B) The synthesis of a polyUb chain and conjugation to a substrate protein is accomplished with a cascade of E1, E2, and E3 enzymes, and the modification is removed with deubiquitinases (DUBs). (C) A polyUb is conformationally heterogeneous, which depends on the covalent Ub linkages and noncovalent Ub-Ub interactions. A covalently linked di-ubiquitin (diUb) can exhibit different relative orientations between the two Ub subunits. Schematic representations of known diUb structures are shown (K48-diUb: 3m3j [34]; K6-diUb:2xk5 [35]; K33-diUb:4xyz [36]; K11-diUb: 2xew [37]; M1-diUb: 2w9n [38]; K63-diUb: 2jf5 [38]; K29-diUb: 4s22 [39]). I44 patches (including residues L8, I44, H68, and V70, shown in red) and I36 patches (including residues I36, L71, and L73, shown in green) are indicated, which are known interacting surfaces. The models of K48-tetraUb and K63-tetraUb are based on references [ 40, 41] . Generated with BioRender.com.

Figure 2

A schematic illustration of stress granule dynamics The SGs, a phase-separated membrane-less organelle, form under certain cellular stress conditions, including RNAs and RNA-binding proteins, such as G3BP1, TDP-43, and TIA-1. It is unclear whether the initial assembly of SGs is ubiquitination-dependent (dashed line), while the disassembly generally involves Ub modification. For example, K63-linkage ubiquitination of G3BP1 promotes its interaction with VCP, as required for the SG disassembly. Moreover, VCP recruits 26S proteasome to facilitate the clearance through the Ub-proteasome system (UPS), in which K48-linkage ubiquitination is involved. Prolonged stress or disruption of proteostasis can lead to the formation of aberrant SGs and insoluble aggregates, whose ultimate removal is mediated by autophagy, a process in which K63-linkage ubiquitination is involved. Generated with BioRender.com.

Figure 3

A schematic diagram showing how ubiquitination can participate in NF-κΒ and Wnt signaling and modulate their phase separation behavior (A) NF-κB complex is responsible for activating many downstream proteins involved in immune and inflammatory responses. NEMO condensates are formed through the non-covalent interactions between anchored polyUb chains and NEMO following the stimulation of TNFα or IL-1β. IKKα and IKKβ are further recruited to the NEMO-polyUb condensates, which leads to the phosphorylation of IκB. Phosphorated IκB is then degraded by the ubiquitin-proteasome system, allowing for the release of NF-κB protein. (B) β-Catenin, a co-activator of transcription factors, is maintained at low levels in the cytoplasm through proteolysis mediated by the GSK3β-AXIN-βTrCP complex. When Wnt proteins bind to LRP5/6 and Fz proteins, Dvl2 can be ubiquitinated by E3 ligases WWP2, which leads to the formation of K63-polyUb-Dvl2 condensate. GSK-3β, AXIN, and βTrCP proteins may also be recruited to the condensate, preventing β-catenin from degradation and allowing β-catenin to be transferred into the nucleus for transcription activation. Generated with BioRender.com.

Figure 4

A schematic diagram showing how ubiquitination is essential for p62-mediated protein quality control The p62 and polyUb undergo co-phase separation; the resulting p62 bodies exist in both cytoplasm and nucleus, albeit with different architectures and functions. Cytoplasmic p62 bodies, consisting of K63-polyUb (predominantly), K48-polyUb, and LC3, are essential to autophagy; the nuclear p62 bodies, containing K48-polyUb (mostly), K63-polyUb, and certain Ub-conjugate enzymes in the interior, as well 26S proteasome at the outside, are important for protein degradation through UPS. Generated with BioRender.com.

Figure 5

A proposed unifying model of ubiquitination in regulating phase separation (A) Schematic representation of Ub modification in the scaffold-client co-phase separation. Taking the UBQLN2 system as an example, its phase separation is mainly driven by the homotypic interactions between UBQLN2 proteins. The presence of Ub monomer or polyUb chains that preferentially adopt compact conformations, such as K11- and K48- tetraUb, would disrupt the UBQLN2 oligomerization owing to the strong interaction between Ub and UBA domain of UBQLN2, which leads to an increased C sat (dashed line) and inhibits phase separation. In contrast, in the presence of polyUb chains that preferentially adopt extended conformations, the C sat is likely decreased (dash-dotted line). (B) A schematic representation that Ub modification promotes phase separation. In the case of p62 bodies, the multivalent interaction between polyUb and p62 allows the phase separation to occur (in this case, the system obtains a large enough associative interaction parameter). Here, neither component alone can phase separate, and they both have to reach a threshold concentration—depending on the phenomenological binding affinity—at a particular cross-section. For more physicochemical details of the cross-interaction-driven co-phase separation, please refer to references [ 161, 162] . Generated with BioRender.com.