Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: Interaction of HPV16E7 and USP7 protein. A Representative immunoblot shows that USP7 binds to HPV16E7. The purified GST fusion proteins of HPV16E7full length, E7 NT and E7 CT were incubated with lysates from cells transfected with Flag-USP7. The levels of Flag-USP7 and GST fusion proteins were analyzed by western blotting. B The bar chart compares the relative levels of USP7 pulled down by different HPV16E7 domains (n = 3). Data were expressed as mean ± standard errors of the means (SEM). ns: not significant, p > 0.05; *p < 0.05. C Immunofluorescent images show the subcellular localization pattern of HPV16E7 and USP7. U-2 OS cells were transfected with pcDNA:HA-HPV16E7 or pcDNA 3.1, followed by incubation with anti-USP7 and anti-HA antibodies. Cellular localization of USP7 and E7 were visualized by fluorescent microscope under ×1000 magnification

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Western Blot, Purification, Incubation, Transfection, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: USP7 extends the half-life of HPV16E7 through de-ubiquitination. A pDONR201:Flag-USP7 or pcDNA 3.1 were transfected into CaSki cells, followed by incubation with cycloheximide for different times as indicated. MG132 (5 μg/ml) was added 2 h before cycloheximide treatment as a control. The cells were harvested and subjected to western blotting analysis. CHX: cycloheximide; KDa: kilodalton. B The protein level curve of E7 protein with or without overpressed USP7. The stability of HPV16E7 was analyzed by the one-phase exponential decay function in GraphPad™ Prism 9. Error bars indicate SEM. C Bar chart comparing the relative levels of ubiquitin, normalized by β-actin (n = 3). Data were expressed as mean ± SEM. D The representative western blotting image shows ubiquitination of HPV16E7. HEK293 cells were transfected with plasmids expressing HA-ubiquitin, HA-HPV16E7 or Flag-USP7 alone, or co-transfected these plasmids together as indicated. The cells were harvested and subjected to co-immunoprecipitation using protein A/G beads. The protein levels of ubiquitin and E7 were analyzed by western blotting. IP immunoprecipitation, IB immunoblotting, WCL whole cell lysates. E The bar graph compares the relative protein level of ubiquitin attached to HPV16E7 (n = 3). Data are expressed as mean ± SEM. *p < 0.05. F CaSki and C-33A cells were transfected with siRNA against control (siCtrl) or USP7 (siUSP7). The cells were harvested and subjected to western blotting analysis

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Transfection, Incubation, Western Blot, Expressing, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: HBX 19818 treatment specifically reduces HPV16E7 stability. A The representative immunoblot shows the effects of USP7 inhibitors upon the protein levels of HPV16E7. CaSki cells were treated with different USP7 inhibitors at low or high concentrations, as indicated; DMSO was used as control. Cell extracts were then analyzed by western blotting. KDa: kilodalton. B The bar chart compares the relative levels of HPV16E7, normalized with β-actin (n = 3). Data are expressed as mean ± SEM; The cell image shows the morphology of the CaSki cells treated with different USP7 inhibitors under ×100 magnification. C The immunoblot shows the protein levels of HPV16E7 at different concentrations of HBX 19818. CaSki cells were treated with different concentrations of HBX 19818 or DMSO as indicated. Total cell lysates were subjected to western blotting with anti-USP7, anti-actin and anti-E7 antibodies. D The bar chart shows the relative levels of HPV16E7 on treatment with different concentrations of HBX 19818; normalized by β-actin (n = 3). Data are expressed as mean ± SEM. E The curve chart indicates the E7 protein levels with increasing concentrations of HBX 19818. The percentage of HPV16E7 protein level was analyzed by the non-linear regression function in GraphPad™ Prism 9. Error bars show SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: The half-life of HPV16E7 is shortened by HBX 19818 through de-ubiquitination. A Immunoblots show protein levels of HPV16E7 at different concentrations of HBX 19818. CaSki cells were transfected with either pcDNA3.1 or pDONR201:Flag-USP7, or were treated with 3 μM HBX 19818, followed by incubation with cycloheximide for different times, as indicated. The cells were harvested and subjected to western blotting analysis. CHX cycloheximide, KDa kilodalton. B The protein level curve of E7 protein with overpressed USP7 or HBX 19818. The stability of HPV16E7 was analyzed by the one-phase exponential decay function in GraphPad™ Prism 9. Error bars show standard errors of the means (SEM). C The bar chart compares the relative protein level of ubiquitin normalized by β-actin (n = 3). Data are expressed as mean ± SEM. D The representative immunoblot image shows ubiquitination of HPV16E7. HEK293 cells were transfected with plasmids expressing HA-ubiquitin, HA-HPV16E7 or Flag-USP7 alone, or these plasmids were co-transfected together, followed by incubation with HBX 19818, as indicated. The cells were harvested and subjected to co-immunoprecipitation using protein A/G beads. The levels of ubiquitin and E7 were analyzed by western blotting. IP immunoprecipitation, IB immunoblotting, WCL whole cell lysates. E The bar chart compares the relative protein level of ubiquitin linked to HPV16E7 (n = 3). F The bar chart compares the relative protein level of USP7 conjugated to HPV16E7 (n = 3). Data are expressed as mean ± SEM. ns not significant, p > 0.05; *p < 0.05

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Western Blot, Transfection, Incubation, Expressing, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: HBX 19818 disrupts the USP7-E7 complex but does not prevent E7-induced pRb degradation. A Immunoblot image shows USP7 binding to E7 in the presence of different concentrations of HBX 19818. Purified GST-HPV16E7 CT protein was incubated with lysates from pcDNA:Flag-USP7-transfected cells with different concentrations of HBX 19818. The levels of Flag-USP7 binding to GST-HPV16E7 CT were analyzed by western blotting. B The bar chart compares the relative level of USP7 bound to HPV16E7 CT (n = 3). Data are expressed as mean ± SEM. C The immunoblot shows the relative pRb levels with or without HBX 19818. HEK293 cells were transfected with plasmids expressing HPV16E7, USP7 or pRb in different combinations as indicated. Transfected cells were then treated with HBX 19818 or DMSO, followed by cell lysis and western blotting analysis. D The bar chart compares the relative level of pRb normalized by β-actin (n = 3). Data are expressed as mean ± SEM

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Western Blot, Binding Assay, Purification, Incubation, Transfection, Expressing, Lysis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis

doi: 10.1007/s00018-023-04941-2

Figure Lengend Snippet: Hypothetical model for USP7-mediated stabilization of HPV16E7 and promotion of carcinogenesis. In the cell nucleus, The E1 ubiquitin-activating enzyme E1 activates ubiquitin molecules through ATP-dependent reaction and the activated ubiquitin is then transferred to the E2 ubiquitin-conjugating enzyme. E3 ubiquitin ligase (e.g. Skp-cullin-F for HPV16E7) attaches the ubiquitin from the E2 enzyme to lysine residues of HPV16E7 to form ubiquitin chains. Subsequently, ubiquitin-tagged E7 will be directed to undergo proteasomal degradation. USP7 removes ubiquitin from HPV16E7, thus protecting E7 from degradation. The binding of HBX 19818 to the ATP pocket of USP7 disrupts its de-ubiquitinase function, thereby preventing the de-ubiquitination of HPV16E7. As a consequence, the ubiquitinated HPV16E7 is then subjected to proteasomal degradation. The HBX 19818-induced destabilization of HPV16E7 leads to the inhibition of E7-mediated carcinogenesis, including cell proliferation, transformation, migration and invasion

Article Snippet: The pDONR201: Flag-tagged USP7 (RRID: Addgene_104959) was kindly provided by Prof. HUEN, Michael Shing Yan; pGEX-2T: HA-HPV16E7 (RRID: Addgene_13634), E7 NT (amino acid 1–52) and E7 CT (amino acid 44–98); and pCMV: HA-HPV16E7 (RRID: Addgene_13686), HA-Rb (RRID: Addgene_58905) and HA-ubiquitin (RRID: Addgene_131258) were kind gifts from Dr. Lawrence Banks.

Techniques: Binding Assay, Inhibition, Transformation Assay, Migration