Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (p97-PROTAC)
- Institute of Medicine, Faculty of Medicine, Universidad Austral de Chile, Chile
- HM CINAC (Centro Integral de Neurociencias Abarca Campal), Hospital Universitario HM Puerta del Sur, HM Hospitales, Spain
- Biotechnological Exploration Laboratory, Health Care Science Faculty, Universidad San Sebastian, Chile
- Technische Universität Berlin, Chair of Bioanalytics, Germany
- Programa de Doctorado en Biomedicina, Facultad de Medicina, Universidad de los Andes, Chile
- Neurodegenerative Diseases Laboratory, Center for Biomedicine, Universidad Mayor, Chile
- Immunoepigenetics Laboratory, Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Chile
- Neuroscience Program, Centro de Investigación e Innovación Biomédica (CiiB) & School of Medicine, Facultad de Medicina, Universidad de los Andes, Chile
- IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Chile
- CETRAM & Faculty of Medical Science Universidad de Santiago de Chile, Chile
- Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Sir James Black Centre, United Kingdom
- Berking Biotechnology SpA, Chile
- Berking Bioscience GmbH, Germany
Figures
Figure 1 with 1 supplement
p97-mediated proteasome degradation.
(A) Schematic representation of p97 presenting ubiquitinated proteins to the proteasome via a UBX domain-containing adaptor (top). The p97-PROTAC system, consisting of a UBX domain fused to a nanobody (Nb) that recruits substrates for p97-mediated segregation, unfolding, and proteasomal-mediated degradation (bottom). (B) p97-PROTAC (UBX-Nb(GFP)) recognizes GFP-tagged proteins at different cellular locations. HeLa cells were seeded on coverslips and co-transfected with UBX-Nb(GFP) and GFP-Coilin, GFP-Emerin, and GFP-ETV1. Cells were fixed and immunostained with anti-myc tag to verify the expression of UBX-Nb(GFP). Colocalization was evaluated using ImageJ/Fiji with the Coloc 2 plugin, obtaining Pearson correlation coefficient (R) values as follows: GFP-Emerin=0.95, GFP-Coilin=0.96, and GFP-ETV1=0.95. (C) Western blot analysis of GFP-Coilin degradation by transfection with p97-PROTAC (UBX-Nb(GFP)). (D) Quantification of C. (E) Western blot analysis of GFP-Emerin degradation by transfection with p97-PROTAC (UBX-Nb(GFP)). (F) Quantification of E. (G) Western blot analysis of GFP-ETV1 degradation by transfection with p97-PROTAC (UBX-Nb(GFP)). (H) Quantification of G, GFP-Coilin: 2 μg ‘p-value’ 0.0011 (**), 4 μg ‘p-value’ 0.0009 (***). GFP-Emerin: 2 μg ‘p-value’ 0.0130 (*), 4 μg ‘p-value’ 0.0059 (**). GFP-ETV1: 2 μg ‘p-value’ 0.0041 (**), 4 μg ‘p-value’ 0.0020 (**). Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 1—source data 1
Raw Western blot images supporting GFP-Coilin, GFP-Emerin, and GFP-ETV1 degradation assays.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig1-data1-v2.zip
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Figure 1—source data 2
Annotated Western blot images indicating protein bands corresponding to GFP-Coilin, GFP-Emerin, and GFP-ETV1 degradation assays.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig1-data2-v2.zip
Figure 1—figure supplement 1
p97-PROTAC sequence, colocalization analysis, and controls confirming UBX-dependent degradation.
(A) Amino acid sequence of human FAF1 UBX domain and synthetic p97 PROTAC chimera. (B–D) Histogram visualization of the colocalization of GFP-Coilin (B), GFP-Emerin (C), or GFP-ETV1 (D) and the Myc-tagged UBX-nanobody targeting GFP in HeLa cells. (E–G) HeLa cells were co-transfected with GFP-Coilin (E), GFP-ETV1 (F) or GFP-Emerin (G) with an empty vector or increasing concentrations of UBX-Nb (Neg.ctrl) vector. Protein degradation was analyzed by western blot. (H–I) Control experiment showing the localization of Myc-UBX-Nb(GFP) in HeLa cells co-transfected with free GFP (H) or empty vector (I).
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Figure 1—figure supplement 1—source data 1
Raw Western blots showing UBX-dependent degradation using an alternative nanobody as a control.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Annotated Western blots showing UBX-dependent degradation using an alternative nanobody as a control.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig1-figsupp1-data2-v2.zip
Figure 2
Targeting liquid-liquid phase separation proteins by a p97-PROTAC.
(A) Strategy for inserting a yellow fluorescent protein (YFP) tag on the N-terminus of the 53BP1 gene in U2OS SEC-C cells using CRISPR/Cas9 D10A. (B) Selected knock-in (KI) YFP-53BP1 clones isolated via flow cytometry. Clones were confirmed via fluorescence microscopy (GE Deltavision Widefield). (C) Super-resolution images obtained with a Delta Vision OMX V4 structured illumination microscope (3D-SIM). (D) Immunofluorescence against 53BP1 (red) and colocalization with YFP-53BP1 in KI cells. Images were obtained using a Delta Vision OMX V4 structured illumination microscope (3D-SIM). (E) Recruitment of the p97-PROTAC UBX-Nb(GFP) (red) to YFP-53BP1 (green) within liquid-liquid phase separation structures. Data were obtained with a high-content CellDiscoverer 7. UBX-Nb(GFP) was detected using its myc-tag. (F) Western blot analysis of YFP-53BP1 degradation by UBX-Nb(GFP) transfection in the KI U2OS cells. (G) Quantification of F, ‘p-value’ 0.0071 (**). Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 2—source data 1
Raw Western blots showing p97-PROTAC–mediated degradation of YFP-53BP1 in KI U2OS cells.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig2-data1-v2.zip
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Figure 2—source data 2
Annotated Western blots indicating YFP-53BP1 degradation in KI U2OS cells transfected with p97-PROTAC.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig2-data2-v2.zip
Figure 3
Endogenous p97 expression in brain tissue.
Immunohistochemistry against p97 in brain tissue sections from nonhuman primates (Nhp) Macaca fascicularis, rat (Sprague-Dawley), and mouse (C57BL6/C). p97 expression was detected in substantia nigra pars compacta (SNpc), hippocampal, and cortical neurons. n=4.
Figure 4 with 1 supplement
Molecular model and degradation activity of p97-PROTAC.
(A) Model representations of the FAF1 UBX domain (purple), UBX-Nb(GFP) (blue), GFP (green), and the p97 hexamer (light gray cartoon with semitransparent molecular surface representation). (B) A magnified view of the model shown in A. (C) GFP monomer was co-transfected with UBX-Nb (GFP) or empty vector in HeLa cells. Protein degradation was analyzed by western blot analysis. (D) Quantification of C, ‘p-value’ 0.0007 (***). (E) GFP monomer was co-transfected with UBX-Nb(GFP) or empty vector in HeLa cells, after 24 hr, the cells were incubated with DMSO or the proteasome inhibitor MG132 (25 µM final concentration) for 4 hr. Protein degradation was analyzed by western blot. (F) Quantification of E, ‘p-value’ DMSO treatment 0.0120 (*), ‘p-value’ MG132 treatment 0.7776 (ns). (G) p97 was silenced by transfection with VCP-siRNA in HeLa cells. Subsequently, the cells were transfected with GFP-Emerin and either the empty vector or UBX-Nb(GFP) vector. Protein degradation was analyzed by western blot analysis. (H) Quantification of GFP-Emerin in UBX-Nb(GFP) cells treated with either siNT control or a VCP-siRNA, ‘p-value’ 0.0114 (*). (I) Quantification of the UBX-Nb(GFP) (myc-tag) in UBX-Nb(GFP) cells treated with either siNT control or a VCP-siRNA, ‘p-value’ 0.0775 (ns). (J) HeLa cells co-transfected with GFP-Emerin and either empty vector or UBX-Nb(GFP); in addition, the cells were treated with the E1 ubiquitin inhibitor PYR-41 (50 µM) for 4 hr at 37°C. Subsequently, total proteins were extracted, and protein degradation was analyzed by western blot. (K) Quantification of J, ‘p-value’ DMSO treatment 0.0009 (***), ‘p-value’ PYR-41 treatment 0.0003 (***). (L) GFP-Emerin was co-transfected with UBX-Nb(GFP) or empty vector in HeLa cells, after 24 hr the cells were incubated with DMSO (as control) or the p97 inhibitor CB-5083 (4 µM final concentration) for 6 hr. Protein degradation was analyzed by western blot using total proteins. (M) Quantification of L, ‘p-value’ DMSO treatment 0.0071 (**), ‘p-value’ CB-5083 treatment 0.0139 (*). Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 4—source data 1
Raw Western blots showing p97-PROTAC–mediated degradation using inhibitors and siRNA controls.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig4-data1-v2.zip
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Figure 4—source data 2
Annotated Western blots showing p97-PROTAC–mediated degradation under inhibitor and siRNA conditions.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig4-data2-v2.zip
Figure 4—figure supplement 1
Validation of p97-dependent degradation and evaluation of PYR41 treatment.
(A) HeLa cells were co-transfected with a vector expressing anti-GFP nanobody fused to a myc-tag (Myc-Nb-anti-GFP) along with either an empty vector or increasing concentrations of p97-GFP. The degradation of Myc-Nb-anti-GFP was analyzed by western blot. (B) Quantification of panel H: 2 μg (p-value = 0.0410, *), 4 μg (p-value = 0.0063, **). (C) HeLa cells were incubated with different concentrations of PYR-41 (a cell-permeable irreversible inhibitor of ubiquitin-activating enzyme E1) for 4 hr. Subsequently, total cellular proteins were extracted, and western blot analyses were performed. Nitrocellulose membranes were incubated with the primary antibodies anti-p53 and anti-ubiquitin antibody. DMSO was used as a control. n=2.
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Figure 4—figure supplement 1—source data 1
Raw Western blots showing p97-dependent degradation and PYR41 treatment effects.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig4-figsupp1-data1-v2.zip
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Figure 4—figure supplement 1—source data 2
Annotated Western blots indicating protein bands for p97-dependent degradation and PYR41 treatment effects.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig4-figsupp1-data2-v2.zip
Figure 5 with 1 supplement
Degradation of Huntingtin wild type and mutant with p97-PROTAC UBX-Nb(GFP).
HeLa cells were transiently co-transfected with HTT GFP-tagged plasmids containing either 23 CAG repeats (EGFP-HTTQ23-wild-type HTT), 74 CAG repeats (EGFP-HTTQ74: mutant HTT), or 24 CAG repeats (EGFP-HTTQ24). (A) Cells were co-transfected with GFP-HTTQ23 and increasing amounts of the p97 PROTAC UBX-Nb(GFP), degradation was determined by western blot analysis. (B) Quantification of A, 2 μg ‘p-value’ 0.0041 (**), 4 μg ‘p-value’ 0.0014 (**). (C) Immunofluorescence showing the recruitment of p97 PROTAC UBX-Nb(GFP) to GFP-HTTQ23. (D) HeLa cells were co-transfected with GFP-HTTQ74 and increasing amounts of the p97 PROTAC UBX-Nb(GFP), degradation was determined by western blot analysis. (E) Quantification of D, 2 μg ‘p-value’ 0.0016 (**), 4 μg ‘p-value’ 0.0003 (***). (F) Immunofluorescence to demonstrate the recruitment of p97 PROTAC UBX-Nb(GFP) to GFP-HTTQ74. Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 5—source data 1
Raw Western blots showing p97-PROTAC–mediated degradation of GFP-HTTQ23 and GFP-HTTQ74.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig5-data1-v2.zip
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Figure 5—source data 2
Annotated Western blots indicating protein bands for p97-PROTAC–mediated degradation of GFP-HTTQ23 and GFP-HTTQ74.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig5-data2-v2.zip
Figure 5—figure supplement 1
HeLa cells were co-transfected with GFP-HTT Q24 and either the p97-PROTAC UBX-Nb(GFP) or an empty vector.
High-molecular-weight aggregates and protein degradation were analyzed by western blot. Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 5—figure supplement 1—source data 1
Raw Western blots showing p97-PROTAC effects on GFP-HTTQ24 degradation and aggregate levels.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Annotated Western blots indicating protein bands for GFP-HTTQ24 degradation and aggregate analysis under p97-PROTAC treatment.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig5-figsupp1-data2-v2.zip
Figure 6
Degradation of α-synuclein with p97-PROTAC.
(A) HeLa cells were co-transfected with a vector expressing αSynuclein mutant A53T fused to GFP (GFP-αSynuclein A53T) and empty or increasing concentrations of UBX-Nb(Syn87). A53T-GFP degradation was determined by western blot. (B) Quantification of A, 2 μg ‘p-value’ 0.0651 (ns), 4 μg ‘p-value’ 0.0020 (**). (C) Cells were co-transfected with a vector expressing untagged α-synuclein mutant A53T and an empty vector or increasing concentrations of UBX-Nb(Syn87). Untagged αSynuclein A53T degradation was determined by western blot using an anti αSynuclein antibody. (D) Quantification of C, 2 μg ‘p-value’ 0.0240 (*), 4 μg ‘p-value’ 0.0210 (*). (E) Quantification of α-synuclein aggregation via Thioflavin T (ThT) fluorescence. Non-transfected cells and cells transfected with GFP-tagged wild-type α-synuclein (pcDNA5 WT αSyn-GFP), either alone or co-transfected with the p97-based PROTACs containing nanobodies against GFP [UBX-Nb(GFP)] or α-synuclein [UBX-Nb(Syn87)], were analyzed for relative ThT fluorescence intensity normalized to total protein concentration (μg/μL). Bars represent mean ± SEM. Asterisks (*) indicate statistically significant differences compared to control (*p<0.05, Dunnett’s post hoc test); ‘ns’ indicates no significant difference. (F) Representative model of p97-PROTAC functioning in the degradation of proteins and protein aggregates. Western blots were quantified and statistically analyzed using a Student’s t-test. p<0.05 compared to controls. n=3.
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Figure 6—source data 1
Raw Western blots showing p97-PROTAC–mediated degradation of GFP-tagged and untagged α-synuclein A53T.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig6-data1-v2.zip
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Figure 6—source data 2
Annotated Western blots indicating protein bands for p97-PROTAC–mediated degradation of GFP-tagged and untagged α-synuclein A53T.
- https://cdn.elifesciences.org/articles/101496/elife-101496-fig6-data2-v2.zip
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Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (p97-PROTAC)
eLife 14:e101496.
https://doi.org/10.7554/eLife.101496
