Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies

Abstract

The 26S proteasome is responsible for regulated proteolysis in eukaryotic cells. Its substrates are diverse in structure, function, sequence length, and amino acid composition, and are targeted to the proteasome by post-translational modification with ubiquitin. Ubiquitination occurs through a complex enzymatic cascade and can also signal for other cellular events, unrelated to proteasome-catalyzed degradation. Like other post-translational protein modifications, ubiquitination is reversible, with ubiquitin chain hydrolysis catalyzed by the action of deubiquitinating enzymes (DUBs), ~ 90 of which exist in humans and allow for temporal events and dynamic ubiquitin-chain remodeling. DUBs have been known for decades to be an integral part of the proteasome, as deubiquitination is coupled to substrate unfolding and translocation into the internal degradation chamber. Moreover, the proteasome also binds several ubiquitinating enzymes and shuttle factors that recruit ubiquitinated substrates. The role of this intricate machinery and how ubiquitinated substrates interact with proteasomes remains an area of active investigation. Here, we review what has been learned about the mechanisms used by the proteasome to bind ubiquitinated substrates, substrate shuttle factors, ubiquitination machinery, and DUBs. We also discuss many open questions that require further study or the development of innovative approaches to be answered. Finally, we address the promise of expanded therapeutic targeting that could benefit from such new discoveries.

Keywords: PROTAC; deubiquitination; proteasome; ubiquitin; ubiquitination.

Fig. 1.

Structure of the 26S proteasome. (A) A cryo-EM reconstruction of 26S proteasome (bottom, EMDB: 1992) showing the base (upper left) and lid (upper right) sub-complexes and their integration into the RP atop of the CP (bottom image). The DUB Rpn11 (green), together with the components displayed in pink (Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn12, and Sem1), form the lid sub-complex (top right), whereas the remaining RP components Rpn1 (beige), Rpn2 (beige), Rpn10 (indigo), Rpn13 (light blue), and Rpt1-Rpt6 (yellow) from the base sub-complex (top left). (B) Exterior (left) and CP cross-section (lower right) from cryo-EM reconstruction of the substrate-engaged proteasome (EMDB: 9045; PDB: 6EF3). The hollow degradation chamber of the CP is apparent in the cross section with the α-ring and β-rings in dark and light grey respectively. At the RP, a substrate (red) extends through the central channel of the ATPase ring (yellow) with an attached ubiquitin (orange) bound to the DUB Rpn11 (green). Density maps for Rpt5 and two α-subunits (α6 and α7) is omitted to show the substrate within the ATPase ring and CP entry. (C) Ribbon diagram of the CP:RP interface depicting the α-ring (grey), the C-terminal small AAA+ subdomains of Rpt1–6 (spanning red to green coloring), and a substrate (red) at the center of the substrate processing channel. The C-terminal HbYX motifs of Rpts docked into inter-subunit cavities of the CP α-ring are circled (blue). This figure was generated by using UCSF Chimera [182], UCSF ChimeraX [183], Adobe Illustrator (Adobe), and Adobe Photoshop (Adobe).

Fig. 2.

The many forms of ubiquitin modifications. (A) Ribbon diagram of ubiquitin (PDB: 1D3Z) highlighting functional sites, including the C-terminal glycine (G76) that is conjugated to substrate proteins or other ubiquitin molecules; lysine residues (K6, K11, K27, K29, K33, K48, and K63) and the N-terminus (M1), which are used to form ubiquitin chains; and the hydrophobic amino acids L8, I44, and V70 (yellow) that are typically used to bind receptors. Nitrogen is displayed in blue. (B) Depiction of ubiquitin chain types, with homotypic chains containing only one linkage type, heterotypic chains with mixed linkages, and branched or forked chains, in which one or more ubiquitin moieties have multiple ubiquitin molecules conjugated. In multiubiquitinated states more than one ubiquitin or ubiquitin chain is attached to a substrate. This figure was generated by using PyMOL (PyMOL Molecular Graphics System, http://www.pymol.org), Adobe Illustrator (Adobe), and Adobe Photoshop (Adobe).

Fig. 3.

Structure and functional domains of proteasome substrate receptors. (A) Domain layout of scRpn1 (top) illustrating the ubiquitin-binding T1 site (navy) and the T2 site (green), which binds the UBL domain of the DUB Ubp6. The scRpn1 toroid structure is displayed on the lower left, highlighting the T1 and T2 sites (PDB: 4CR2). An expanded ribbon diagram of K48-linked diubiquitin bound to the T1 site is shown on the lower right (PDB: 2N3V). In (A) and (B) the proximal (dark orange) and distal (light orange) ubiquitin moieties are displayed with the K48 linkage site in stick representation (oxygen, red; nitrogen, blue). (B) Domain layout for hRpn10 (top) illustrating the proteasome-binding VWA domain (grey), ubiquitin-binding UIMs and interhelical region (blue), and UBE3A-binding RAZUL domain (cyan). Ribbon diagrams of the VWA domain (lower left), a snapshot of the dynamic UIM region bound to K48-linked diubiquitin with dashed arrows symbolizing flexibility (middle), and the RAZUL:UBE3A AZUL complex (lower right) are displayed. In the AZUL domain, Zn is shown as a blue sphere with coordinating cysteine sulfur atoms in yellow. PDB 2X5N, 2KDE, and 6U19 were used to generate this figure. (C) Domain layout of hRpn13 (top) highlighting the ubiquitin and proteasome binding Pru domain (blue) and UCHL5-binding DEUBAD domain (grey). When free of a binding partner, the two domains interact, as demonstrated in the central ribbon diagram. This structural image has omitted the interdomain region, which is intrinsically disordered. The structure of the Pru domain bound to an extended, dynamic form of K48-linked diubiquitin and 14-amino acid C-terminal region of Rpn2 (gold) is displayed on the lower left. Proximal ubiquitin (orange) is shown bound to the Pru domain loops. The dynamic distal ubiquitin (yellow, eight conformers displayed) exhibits limited order that is defined by interactions at the inter-ubiquitin linker region. A hydrogen bond between the G76 of the distal ubiquitin and K103 of the Pru domain contributes to hRpn13’s preference for K48-linked ubiquitins. The structure of the hRpn13 DEUBAD domain complexed with UCHL5 (green) and a suicide ubiquitin variant (orange) is displayed on the lower right. The DEUBAD domain splits to wrap around UCHL5. PDB 6UYI, 2KR0, and 4WLR were used to generate this figure. (D) Hypothetical model of the ubiquitin-bound 26S proteasome, highlighting the ubiquitin receptors Rpn1 (beige) with T1 (navy) and T2 (green) sites, Rpn10 with the VWA domain (cyan) and UIMs (blue cartoon), and Rpn13’s Pru domain (blue). The ATPase ring, lid (except for Rpn11), Rpn2, Rpn11, and CP are colored yellow, pink, beige, grey, and light green, respectively. Ubiquitin moieties are displayed as yellow or orange ribbon diagrams with the linkage site rendered in stick representation (oxygen, red; nitrogen, blue). This model illustrates how a ubiquitin chain could extend from Rpn10 to Rpn13 and along Rpn1. In addition to the two distinct ubiquitin-chain pathways displayed here, Rpn10’s UIMs or Rpn13’s Pru could bind to different ubiquitin chains of a substrate or alternatively, the ubiquitin chain could bind to just one receptor. The distribution of ubiquitins to the various ubiquitin-binding sites is expected to depend on the number of attached ubiquitin chains, as well as their length and linkage type. PDB 6WJD, 6UYI, 2N3V, 1D3Z, and EMDB 21691 were used to generate this figure. This figure was generated by using UCSF Chimera [182], PyMOL (PyMOL Molecular Graphics System, http://www.pymol.org), Adobe Illustrator (Adobe), and Adobe Photoshop (Adobe).

Fig. 4.

ATPase ring gymnastics. (A) Structural motifs of the proteasome ATPase ring. Left: A top view of the EM density for the Rpt1-Rpt6 hexamer in the s4 state (EMDB: 9045), with the central channel of ATPase ring labeled. Middle: ATPase ring displayed as a ribbon diagram (PDB: 6EF3) rotated 90° relative to the view on the left and highlighting the N-terminal coiled coils, N-ring, ATPase motor ring, and HbYX motifs. Right: ribbon diagram structure of Rpt5 and Rpt6 (PDB: 6EF3) to illustrate the OB fold, Pore-1 loop (red), and nucleotide (oxygen, red; nitrogen, blue; phosphorus, orange). (B) Cutaway representations of the 26S proteasome in s1 (apo, EMDB: 3534) and s4 (engaged, EMDB: 3537) conformations, showing the different position of Rpn11 and change in alignment of the N-ring, ATPase motor ring, and CP. The central channel through the N-ring and ATPase motor ring is indicated by a dashed orange line. (C) Conformational switching of the human 26S proteasome between a ground state (S_A, PDB: 5VFS) and substrate processing state (S_D, PDB: 5VFP), with the CP aligned. During the transition from S_A to S_D, hRpn10’s VWA domain rotates by ~30° towards the hRpt4/hRpt5 coiled coil. The color scheme in panels (B) and (C) follows that in Fig. 1B and Fig. 3D. This figure was generated by using UCSF Chimera [182], UCSF ChimeraX [183], Adobe Illustrator (Adobe), and Adobe Photoshop (Adobe).