Unstructured Biology of Proteins from Ubiquitin-Proteasome System: Roles in Cancer and Neurodegenerative Diseases

Abstract

The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure-function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases.

Keywords: cancer; intrinsically disordered proteins; molecular recognition features; neurodegenerative diseases; protein degradation; protein misfolding; ubiquitin-proteasome system.

Figure 1

Schematic representation of the ubiquitin proteasomal system. Ubiquitination is an ATP-dependent process performed by three enzymes: E1 (Ub-activating) enzyme, E2 (Ub-conjugating) enzyme, and E3 (Ub-ligase) enzyme. The DUBs, such as ataxin-3, modify the polyubiquitinated chain, to confirm accurate recognition of the misfolded proteins by the 26S proteasome. This covalent modification of misfolded protein targets them to multicatalytic protease complex, the 26S proteasome. Ubiquitination is reversed by DUBs and disassembles polyubiquitin chains. DUBs such as USP7, UCHL1, and ataxin-3 also control and maintain free Ub molecules in the cell. UCHL1 modifies newly translated protein and maintains a pool of mono-Ub. The polyubiquitinated misfolded protein can bind either to the Ub receptor of the 19S regulatory complex or to an adaptor protein that consists of both poly-Ub binding and proteasome binding domain [27]. Once misfolded protein binds to proteasome, the unfolding of the misfolded protein occurs by ATPases followed by removal of the poly-Ub chain by proteasome-associated DUBs and further translocation and degradation of unfolded protein in central proteolytic chamber occurs. Excessive degradation of cell-cycle-regulatory proteins such as p21 and p27 and reduced degradation of mutant p53 leads to a continuous cell cycle of cancer cells and tumor progression leads to the development of cancer [28]. Additionally, impairment in function of 26S proteasome, ubiquitinating enzymes, and DUBs can lead to nerve cell death and the progression of neurodegenerative diseases. Ub: Ubiquitin, E1: Ub-activating enzyme, E2: Ub-conjugating enzyme, E3: Ub-ligase enzyme.

Figure 2

Evaluation of the overall disorder status of 15 UPS proteins associated with human diseases. Here, a 2D disorder plot presents the PPID_{PONDR VLXT} vs. PPID_{PONDR VSL2} dependence.

Figure 3

Evaluation of the intractability of 15 UPS proteins associated with human diseases by STRING platform. (A) Network of the inter-set PPI interactions (15 subunits, highest confidence level of 0.9). (B) PPI network centered at 15 UPS proteins associated with human diseases (515 proteins, highest confidence level of 0.9).

Figure 4

Intrinsic disorder in ubiquitinating enzymes E1 (UBA1), E2 (UBE2R2) and E3 (STUB1). (a) ubiquitin-activating enzyme E1 (UniProt ID: P22314), (a1) crystal structure of the E1 enzyme (PDB ID: 6DC6). (b) ubiquitin-conjugating enzyme E2 (UniProt ID: Q712K3), (b1) crystal structure of E2 enzyme having residues 1–202 (PDB ID: 6NYO). (c) E3 ubiquitin ligase (UniProt ID: Q9UNE7), (c1) crystal structure of E3 ubiquitin ligase (PDB ID: 4KBQ). In Plots (a–c), the outputs of PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred are represented by black, orange, blue, yellow, and purple lines, respectively. Mean disorder profile, calculated by averaging the outputs of five predictor-specific per-residue disorder profiles, is depicted by olive color. Light-olive shadow around the mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows error distribution for PONDR^® FIT. In (a1), crystal structure of the E1 enzyme (PDB ID: 6DC6) is represented in faded blue color, disordered residues are shown in salmon pink color, and MoRF residues identified by MoRFCHiBi_Web server (1048–1057) are shown in grey color. In (b1), E2 enzyme (1–202 length with missing residues 1–5 and 193–202 at N- and C-terminal, respectively) is shown with Ubiquitin-60S ribosomal protein L40 (RPL40A; orange color). In (c1), Hsc70 Lid-Tail domains (orange color) in complex with E3 ubiquitin ligase (which is represented in faded blue color); disordered residues in E3 are shown in salmon pink color. In (a2,b2,c2), functional disorder profiles, MoRFs, and PTMs in E1, E2, and E3 enzymes using D2P2 server have been shown.

Figure 5

Analysis of intrinsic disorder predisposition in polyubiquitin-B (UBB) and structural characterization of mature ubiquitins. (a) Disorder analysis of human polyubiquitin-B (UniProt ID: P0CG47). (b) Crystal structure of chain B of Polyubiquitin-B (PDB ID: 6FDK). (c) Crystal structure of Chain D of Polyubiquitin-B (PDB ID: 6BYH). (d) Crystal structure of Chain A of Polyubiquitin-B (PDB ID: 4XOF). In Plot (a), the disorder profile obtained forms a set of disorder predictors such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT and IUPred, represented by black, orange, blue, yellow, and purple curves respectively. Mean disorder profile, which was calculated from average of five predictor-specific per-residue disorder profiles, is shown in olive color. Predicted disorder scores above 0.5 are considered as disordered residues/regions. The light-olive shadow around the mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows the error distribution for PONDR^® FIT. In Plot (b), a structure of Ub protein (faded blue) in complex with Chlamydia trachomatis effector protein Cdu1 (orange color) is represented. In Plots (b), (c), and (d), Ub is shown in faded blue color, and disordered residues are shown in salmon pink color. The position of MoRFCHiBi_Web-server-identified MoRFs (residues 40–50, shown in PDB ID:6FDK, and residues 192–202, shown in PDB ID: 4XOF) are represented by grey color. (e) Functional disorder profile of the UBB protein, using the D2P2 server, is shown.

Figure 6

Intrinsic disorder predisposition and structural characterization of human UBQLN1 and UBQLN2. (a) Disorder profile of human UBQLN1 (UniProt ID: Q9UMX0). (b) Disorder profile of UBQLN2 (UniProt ID: Q9UHD9) (a1) NMR solution structure of the N-terminal UBL domain (residues 34–112) of UBQLN1 (PDB ID: 2KLC). (a2) NMR solution structure of the C-terminal UBA domain (residues 541–586) of UBQLN1 (PDB ID: 2JY5). (b1) NMR solution structure of the N-terminal UBL domain (residues 1–103) of human UBQLN2 (PDB ID: 1J8C). In Plots (a) and (b), intrinsic disorder profiles generated by disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT and IUPred, are shown by black, orange, blue, yellow, and purple curves respectively. Mean disorder profile is calculated from the average of five predictor-specific per-residue disorder profiles, represented by the olive color curve. Predicted disorder scores above 0.5 are considered disordered residues/regions. The light-olive shadow around the mean curve represents error distribution for mean. The light-yellow shadow around PONDR^® FIT curve shows error distribution for that predictor. In (a1,a2,b1), UBQLN 1 and 2 are represented by faded blue color, and disordered residues are shown in salmon pink color. The position of the MoRF region (residues 34–39) recognized by the MoRFCHiBi_web server in UBQLN1 is represented in (PDB ID: 2KLC), and MoRF region (residues 10–38) in UBQLN2 are represented in (PDB ID: 1J8C) as MoRFs lying in the IDP region by faded green color. (a3,b2) Functional disorder profile of UBQLN1 and UBQLN2 proteins, respectively, using the D2P2 server, is shown, depicting PTM sites.

Figure 7

Intrinsic disorder predisposition and structural characterization of UCHL1 and UCHL5. (a) Disorder profile of human UCHL1 (UniProt ID: P09936) (b) Disorder profile of human UCHL5 (UniProt ID: Q9Y5K5) (a1) Crystal structure of UCHL1 (PDB ID: 4JKJ). (b1) Crystal structure of UCHL5 (PDB ID: 4UEL). In (a,b), intrinsic disorder profiles obtained from disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT and IUPred, are depicted by black, orange, blue, yellow, and purple lines respectively. Mean disorder profile, calculated from the average of five predictor-specific per-residue disorder profiles, is represented by the olive color. Predicted disorder scores above 0.5 are considered disordered residues/regions. The light-olive shadow around the mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows the error distribution for PONDR^® FIT. In (a1,b1), UCHL1 and UCHL5 are represented by faded blue color; in Plot (b1), the DEUBAD domain of the RPN13 protein and Ub in complex with UCHL5 are shown in orange color. Disordered residues are shown in salmon pink color. In (a2,b2), the functional disorder profile of UCHL1 and UCHL5 proteins using the D2P2 server have been shown.

Figure 8

Intrinsic disorder predisposition and structural characterization of human USP7 and USP14. (a) Disorder profile of human USP7 (UniProt ID: Q93009). (b) Disorder profile of human USP14 (UniProt ID: P54578) (a1) crystal structure of USP7 (PDB ID: 4YOC). (b1) Crystal structure of USP14 (PDB ID: 4GJQ). In Plots (a) and (b), disorder profiles generated by sets of disorder predictors such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred are depicted by black, orange, blue, yellow, and purple curves respectively. The mean disorder profile calculated from the average of five predictor-specific per-residue disorder profiles is shown by the olive color curve. Predicted disorder scores above 0.5 are considered disordered residues/regions. The light-olive shadow around mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows the error distribution for PONDR^® FIT. In Plots a1 and b1, USP7 and USP14 are represented by faded blue color; in Plot (a1), human DNA (cytosine-5)-methyltransferase 1 (DNMT1) complexed with USP7 is shown in orange color. Disordered residues are shown by the salmon pink color. In Plot (a1), the positions of MoRFs (residues 1077–1082) predicted by the MoRFCHiBi_web server are shown by grey color in USP7 (PDB ID: 4YOC). (a2,b2) depict the PTM sites and MoRF regions obtained from the D2P2 server.

Figure 9

Intrinsic disorder predisposition of ataxin-3. (a) Intrinsic disorder profile generated for ataxin-3 (UniProt ID: P54252) by a set of per-residue disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred. (b) Crystal structure of the Josephin domain of ataxin-3 (PDB ID: 2AGA). (c) NMR solution structure of the tandem UIM domain of ataxin-3 (PDB ID: 2KLZ). In Plot (a), disorder profiles generated by set of disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred, are depicted by black, orange, blue, yellow, and purple curves respectively. A mean disorder profile calculated from average of five predictor-specific per-residue disorder profile is shown by the olive color curve. Predicted disorder scores above 0.5 are considered as disordered residues/regions. The light-olive shadow around the mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows the error distribution for PONDR^® FIT. In Plots (b) and (c), ataxin-3 is represented by a faded blue color; disordered residues are shown by a salmon pink color. In (b), the position of MoRFs (residues 56–65) predicted by the MoRFCHiBi_web server is shown by a gray color (PDB ID: 2AGA). In (c), the position of MoRFs (residues 246–255) predicted by the MoRFCHiBi_Web server is represented as MoRFs lying in IDPRs by faded green color (PDB ID:2KLZ). (d) The D2P2 server-based functional disorder profile is shown.

Figure 10

Intrinsic disorder predisposition of ADRM1. (a) Intrinsic disorder profile generated for ADRM1 (UniProt ID: Q16186) by a set of per-residue disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred. (b) D2P2 server-based functional disorder profile for ADRM1. (c) Solution NMR structure of the ADRM1 with 20 different conformations (PDB ID: 2KR0). In Plot (a), disorder profiles generated by sets of disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred, are depicted by black, orange, blue, yellow, and purple curves, respectively. The mean disorder profile, calculated from the average of five predictor-specific per-residue disorder profiles, is shown by the olive color curve. The predicted disorder score above 0.5 are considered as disordered residues/regions. The light-olive shadow around mean curve represents the error distribution for the mean. The light-yellow shadow around PONDR^® FIT curve shows error distribution for PONDR^® FIT. In (c), ADRM1 is represented by the faded blue color, and disordered residues are shown by the salmon pink color.

Figure 11

Intrinsic disorder predisposition of human PSMD2 (a), PSMD4 (b), and PSMD14 (c). Plots show the intrinsic disorder profile generated by a set of per-residue disorder predictors, such as PONDR^® VSL2, PONDR^® VL3, PONDR^® VLXT, PONDR^® FIT, and IUPred. The corresponding outputs are depicted by black, orange, blue, yellow, and purple curves respectively. Mean disorder profile is calculated from the average of five predictor-specific per-residue disorder profiles and shown by the olive color curve. Predicted disorder scores above 0.5 are considered disordered residues/regions. Light-olive shadow around the mean curve represents the error distribution for the mean. The light-yellow shadow around the PONDR^® FIT curve shows the error distribution for that predictor. (d) portrays the cryo-EM structures of substrate-engaged human 26S proteasome in seven different conformational states (PDB ID: 6MSB); all three reported proteins PSMD2 (blue), PSMD4 (wine) and PSMD14 (olive) are shown in this complex. D2P2 server-based functional disorder profiles are shown in (a1,b1,c1).