Article Figures & Data
Figures
- Figure 1. Activation of the 26S proteasome by FAT10 and NUB1L.
(A) A native gel overlay assay was performed with human 26S proteasome in presence or absence of FAT10 and NUB1L. Samples were initially incubated for 15 min at 37°C with a 10-fold molar excess of NUB1L and FAT10 as compared to the 26S proteasome, before gel electrophoresis was performed. The overlay assay was performed with Suc–LLVY–AMC for 30 min at 37°C. The gel was then developed under UV light. The marker was a mix out of 20S and 26S proteasomes. (B) Z–GGL–AMC hydrolysis was monitored in an activity assay. Equal amounts of 26S proteasome (5 nM) were incubated with an excess of NUB1L and FAT10 (500 nM) or ubiquitylated E6AP. The chymotrypsin-like activity was measured with Z–GGL–AMC (10 μM) at 37°C. The fluorescence intensity was measured over a period of 90 min. Proteasomal activity was expressed in relation to 26S proteasome without any stimulant. (C) The activity assay was performed as in (B) but in the presence or absence of 1 μM proteasome inhibitor MG132. (D) The activity assay, as described in (B), was performed with equal amounts of 20S and 26S proteasomes (5 nM). (E) The activity assay as described in (B) was performed with equal concentrations of ADP, ATP, and the non-hydrolysable ATPγS (0.5 mM) in the presence or absence of FAT10 and NUB1L. (F) Shows the same experiment as performed in (E) but the percentage of activity was always calculated between the corresponding control (black) and the 26S proteasome in presence of FAT10 and NUB1L (grey). (G) The activity assay was performed as described in (B) but in addition to Z–GGL–AMC (10 μM) to determine the chymotrypsin-like activity, the trypsin-like activity with LRR–AMC (10 μM), and the caspase-like activity with Ac–nLPnLD–AMC (10 μM; nL = norleucine) was measured. (H) The activity assay was performed as described in (B) using the native FAT10 and the stabilized form of FAT10, FAT10c0c134L, in the presence or absence of NUB1L. Statistical analysis was performed using an unpaired t test (ns, not significant: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001).
Source data are available for this figure.
Source Data for Figure 1[LSA-2022-01463_SdataF1.xlsx]
- Figure 2. The degradation of FAT10 by open gate and WT proteasome.
(A) A Western blot was performed with samples of two cycloheximide chase experiments. On the left side, WT S. cerevisiae was transfected with HA–FAT10 Met vector, and the degradation of FAT10 was monitored over the period of 4 h. On the right side, the 20S proteasome open-gate yeast mutant α3∆N was transfected with the same vector. The degradation of HA–FAT10 was increased because of the open-gate state of the 26S proteasome. (B) The experiment is the same as in (A), except for the co-transfection of both yeast types with HA–NUB1L in a Leu vector in addition to the HA–FAT10 Met vector. In the open-gate mutant, the degradation of HA–FAT10 was not accelerated by NUB1L in contrast to the WT yeast (when compared with HA–FAT10 degradation in panel (A)).
- Figure 3. Dissection of domains in FAT10 and NUB1L for activation of the 26S proteasome.
(A) The activity assay was performed as described in Fig 1B. In addition, the 26S proteasome was incubated with equal amounts of USE1 and the USE1–FAT10 conjugate (500 nM). Only FAT10 and FAT10ylated USE1 together with NUB1L led to an increase in proteasomal activity. (B) Equal amounts of indicated known NUB1L interaction partners (500 nM) were incubated with NUB1L and the 26S proteasome. Only FAT10 showed the activating effect together with NUB1L. (C) Recombinant NUB1L variants lacking either the three UBA or the UBL domain were incubated with the 26S proteasome in the presence or absence of FAT10. Only full-length NUB1L had the ability to lead to an activation of the 26S proteasome. (D) The two FAT10–UBLs were incubated alone or together with NUB1L. Only full-length FAT10 was able to activate the 26S proteasome together with NUB1L. (E) Recombinant NUB1L–UBL–GFP–cytb containing the UBL domain of NUB1L only, FAT10–GFP and FAT10–GFP–cytb fusion proteins were tested in equal amounts for their ability to activate the 26S proteasome, as indicated. Although conjugated FAT10–GFP–Cytb and FAT10–GFP are both suitable to activate the 26S proteasome, the UBL domain in NUB1L–UBL–GFP–Cytb is not enough for the activation. (F) A NUB1–p21–FAT10 fusion protein was incubated with the 26S proteasome to test whether NUB1L and FAT10 need to be covalently linked, for example, by a flexible linker (p21) for activation of the 26S proteasome. Activation by FAT10 and NUB1L served as positive control. Statistical analysis was performed using an unpaired t test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001).
Source data are available for this figure.
Source Data for Figure 3[LSA-2022-01463_SdataF3.xlsx]
- Figure 4. FAT10 increases the affinity of NUB1L for binding to the 26S proteasome.
(A) Western blot of a native gel, displaying the interaction of FAT10 and NUB1L with the 26S proteasome. NUB1L alone, and FAT10 and NUB1L together are capable of interfering with the non-covalent binding of USP14 to the 26S proteasome. (B) Octet-binding curve of NUB1L-bound GST–FAT10 (red), NUB1L and GST (light blue), NUB1L only (dark blue), and GST–FAT10 (green) for membrane-bound His6–RPN1. The binding of NUB1L to RPN1 increases markedly when GST–FAT10 is bound by NUB1L as is also documented by the calculated dissociation constants Kd shown in the table below. (C) A pulldown (PD) was performed with GST–NUB1L. On the left side, FAT10 was incubated with GST–NUB1L and competed with increasing amounts of the UBL domain of NUB1L, fused to GFP–Cytb (named as NUB1L–UBL–GFP–Cytb): lane 1, 1:0; lane 2, 1:1; lane 3, 1:2; lane 4, 1:4; and lane 5, 1:8. At a ratio of 1:2, the NUB1L–UBL–GFP–Cytb was able to bind to NUB1L and to displace FAT10–GFP–Cytb. On the right side, the experiment was changed and NUB1L–UBL–GFP–Cytb had to compete with increasing concentrations of FAT10. In this case, a 1:1 ratio was sufficient to displace NUB1L–UBL–GFP–Cytb from GST–NUB1L and results in both proteins being bound to GST–NUB1L.
- Figure S1. FAT10 binding to RPN10.
Octet-binding curve of NUB1L∆UBL, GST–FAT10 alone, and GST–FAT10 together with NUB1L∆UBL to the His6–RPN10 bound sensor. In contrast to Fig 4B, the binding affinity of GST–FAT10 to His–RPN10 was not significantly changed by NUB1LΔUBL.
- Figure 5. USP14 independence of 26S proteasome activation by FAT10 and NUB1L.
(A) A GST pulldown (PD) was performed using 20 μl GST–RPN1 incubated with ∼10 ng NUB1L. NUB1L was then eluted by increasing amounts of USP14 (10 ng (1), 20 ng (2), 50 ng (3), and 100 ng (4)); in lane 5, GST served as negative control. Lanes 6–10 show the same experiment with additional 5 ng FAT10. An increase of bound NUB1L to RPN1 is visible in presence of FAT10 (compare lanes 1–4 to 6–9, second gel from top: IP: GST, WB: NUB1L). (B) The USP14 WB shows the abundance of USP14 in purified 26S proteasome from erythrocytes, the mouse fibroblast line C4, and the USP14 knockout mouse fibroblast line. PSMB5 was used as loading control. (C) A fluorogenic peptide (Z–GGL–AMC) based activity assay was performed as described in the legend to Fig 1B. The 26S proteasomes purified from the USP14 proficient fibroblast line C4 and a USP14 deficient fibroblast line were equally activated by FAT10 + NUB1L. Statistical analysis was performed using an unpaired t test (*P ≤ 0.05). (D) Hypothetical scheme in which FAT10 resolves NUB1L dimers by binding to the UBA domains of NUB1L. Thereby, the UBL domain of NUB1L gets free to bind to RPN1 and replace USP14. Consequently, the C-domain of FAT10 is free to bind to RPN10 and activate the 20S gate opening.
Source data are available for this figure.
Source Data for Figure 5[LSA-2022-01463_SdataF5.xlsx]
- Figure S2. The degradation of poly-ubiquitylated proteins by the 26S proteasome in vitro was affected by FAT10 and NUB1L.
(A) An in vitro degradation assay was performed with ubiquitylated SIC1. The mix of poly-ubiquitylated SIC1, NUB1L, FAT10, and 26S proteasome was incubated for 20 min at 37°C. At time points 0, 5, 10, and 20 min samples were taken, and a Western blot against poly-ubiquitin was performed. On the right side, the samples were incubated with an excess of NUB1L and FAT10. The bar graph below shows the quantification of the Western blot ECL signals. Time point 0 min was set to unity, and all other values were calculated accordingly. (B) An in vitro degradation assay was performed with radioactively labeled ubiquitylated p53. The mix out of poly-ubiquitylated p53, NUB1L, FAT10, and 26S proteasome was incubated as described above. The first samples show degradation of p53 by autoradiography under conditions without NUB1L and FAT10. On the right side, the samples were incubated with an excess of NUB1L and FAT10 slowing down ubiquitin–p53 degradation. The bar graph shows the quantification of the autoradiography signals. Time point 0 min was set to unity and all other values were calculated accordingly.
Source data are available for this figure.
Source Data for Figure S2[LSA-2022-01463_SdataFS2.xlsx]
- Figure S3. The binding affinity of RAD23 and NUB1L to RPN1 mutants.
(A) Octet-binding curve of His6–RPN1 WT and mutants His6–RPN1 T1, His6–RPN1 T2, and His6–RPN1 T1 + T2 to GST–NUB1L bound to the Octet sensor. (B) Octet-binding curve of His6–RPN1 WT and GST to GST–RAD23 bound to the Octet sensor. (C) Octet-binding curve of His6–RPN1 T1 and GST to GST–RAD23 bound to the Octet sensor. (D) Octet-binding curve of His6–RPN1 T2 and GST to GST–RAD23 bound to the Octet sensor. (E) Octet-binding curve of His6–RPN1 T1 + T2 and GST to GST–RAD23 bound to the Octet sensor. (F) Pulldown using 20 μg of GST–NUB1L and adding ∼10 μg of His6–RPN1 WT (1), His6–RPN1 T1 (2), His6–RPN1 T2 (3), and His6–RPN1 T1 + T2 (4). Lanes 5–8 show the excessive amount of the RPN1 variants in the supernatant. (G) Pulldown using 20 μg of GST–hRAD23 (HR23) and ∼10 μg of His6–RPN1 WT (1), His6–RPN1 T1 (2), His6–RPN1 T2 (3), and His6–RPN1 T1+T2 (4). Lanes 5–8 show the excessive amount of the RPN1 variants in the supernatant.
