Fig 1: USP7 regulates misfolded mutant SOD1 protein levels through the transcription factor SMAD2. (A) The SMAD transcriptional activity was increased in HEK293 cells upon USP7 knockdown (Left) or inhibition of USP7 by the small-molecule drug HBX41108 at 2 μM (Right), as measured by the SMAD response element (SMAD-RE)–mediated luciferase activity assay (n = 3; *P < 0.05, ****P < 0.0001). (B) The protein levels of SMAD2, but not those of SMAD3, were increased upon USP7 knockdown (n = 3; **P < 0.01). (C) Endogenous SMAD2 was coimmunoprecipitated when Flag-USP7 expressed in HEK293 cell was pulled down by the anti-Flag antibody but not the IgG control. (D) Immunoblot analysis of SOD1G85R in cells with USP7 or SMAD2 knockdown, or double knockdown, indicated that loss of SMAD2 abolished the effect of USP7 on the regulation of SOD1G85R levels in both the supernatant and pellet fractions (n = 4; *P < 0.05, **P < 0.01). Error bars indicate ± SEM.
Fig 2: USP7 deubiquitinates NEDD4L, the E3 ligase for SMAD2. (A) Immunoblots and quantification of endogenous NEDD4L in HEK293 cells with USP7 or control knockdown (n = 3; *P < 0.05). (B) Endogenous NEDD4L was coimmunoprecipitated when Flag-USP7 expressed in HEK293 cells was pulled down by the anti-Flag antibody but not the IgG control. (C) In the in vitro deubiquitination assay, V5-NEDD4L expressed in HEK293 cells was immunoprecipitated and used as a substrate for incubation with purified GST-USP7 or GST. The presence of USP7 significantly decreased the levels of ubiquitinated NEDD4L protein (n = 3; *P < 0.05). Error bars indicate ± SEM. (D) In the in vivo deubiquitination assay, cell lysates expressing Flag-USP7 variants, V5-NEDD4L, and HA-Ub and were incubated with anti-V5 antibody to pull down NEDD4L, and the immunoprecipitates were subjected to analysis of ubiquitination by immunoblotting. C223S is a catalytic mutant of USP7, and GUS is a control.
Fig 3: Role of the USP7–SMAD2 pathway in neurons and patient tissues. (A) Mouse neurons were transduced with SOD1G85R or SOD1WT HSVs and treated with vehicle (DMSO) or the USP7 inhibitor HBX41108. (Scale bar, 150 μm.) Live cells were loaded with the calcein AM dye to determine cell viability (Left). Quantification of the cell viability (Right; n = 8; n.s., nonsignificant; *P < 0.05). (B) Representative Western blotting of human spinal cord tissue lysates derived from ALS patients and controls show up-regulation of SMAD2 in the patients’ tissues (Left). Quantification of the SMAD2 protein levels (Right; n = 8 for control and n = 24 for ALS; **P < 0.01). Error bars indicate ± SEM.
Fig 4: A reduction in the USP7 function increases clearance of misfolded proteins. (A) Western blot analysis of cell lysates derived from mock (CTRL) or USP7 knockdown in HEK293 cells expressing SOD1G85R. USP7 knockdown reduced the SOD1G85R proteins in both the supernatant (S) and pellet (P) fractions, without affecting the level of endogenous WT SOD1 (SOD1WT). Quantification of SOD1WT and SOD1G85R protein levels by immunoblotting is shown (n = 3; ***P < 0.001, ****P < 0.0001; n.s., nonsignificant). (B) A small-molecule inhibitor of USP7, HBX41108, reduced the level of misfolded SOD1G85R protein, but not that of SOD1WT protein in HEK293 cells. A dose-dependent effect on the level of SOD1G85R protein with increasing concentrations of HBX41108, as compared to the vehicle control (DMSO), was observed in the supernatants of the cell lysates (n = 3; n.s., nonsignificant; *P < 0.05). (C) Western blots of a representative cycloheximide chase experiment to determine the SOD1 protein half-life in the USP7 knockdown cells versus controls (Left). Quantification of the SOD1G85R clearance as analyzed by immunoblotting is shown (Right). The graph indicates the relative band intensity of SOD1G85R at each chase time point in the USP7 knockdown cells versus controls (n = 3; *overall P < 0.05 for the three time points after 0 h). Error bars indicate ± SEM.
Fig 5: The knockdown of Drosophila Usp7 suppresses neurotoxicity induced by mutant SOD1 or TDP-43. (A) Chart of the climbing (negative geotaxis) assay in adult Drosophila expressing human SOD1A4V in motor neurons (Left). Quantification of the climbing ability of Drosophila expressing human SOD1A4V in motor neurons, together with gene-specific RNAi against Usp7 (34708), Smox (26756), Babo (40866), or control RNAi (Right; n = 8 independent groups; **P < 0.01). (B) Reduction in Usp7 by RNAi (34708) strongly suppresses the eye degeneration phenotypes in TDP-43M337V strains, while reduction in Smox (RNAi 26756) or Babo (RNAi 40866) worsens the eye degeneration phenotypes, when compared with the control Luc RNAi strain (Left). (Scale bar, 100 μm.) Quantification of pigment content in adult eyes confirms the protection against degeneration by a loss of Usp7 in TDP-43M337V strains (Right). The measurements represent three independent groups, each containing fly heads from two males and two females (n = 3; **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars indicate ± SEM.
Supplier Page from OriGene Technologies for USP7 Human shRNA Plasmid Kit (Locus ID 7874)