Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: In the presence of SIP, the levels of N-terminal HTT decrease and mutant HTT aggregates decrease. a SIP decreases the expression of proteins encoded by exon 1 of wt HTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP. b SIP decreases the aggregation of proteins encoded by exon 1 mHTT in the cellular HD model. HEK293T cells co-transfected with plasmids that are indicated in the figure: control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP. The left parts of panels a and b were imaged under a fluorescent microscope. Images in the right parts of panels a and b were taken under transmitted light. Scale bar = 1 µM. The size and number of mHTT aggregates (e1HTT-72Q) are presented in panels b ’ and b ”, respectively. Red color signals of e1HTT-72Q were measured as the number of pixels per aggregate b ’ and show the average aggregate size measured for each of the analyzed images. c , d Western blot (WB) analysis of HEK293T cells that co-expressed e1HTT-25Q c or e1HTT-72Q d and wt SIP or control pEntry plasmids. c , d Graphs show densitometric analysis of western blotting bands for the indicated proteins using GAPDH abundance for normalization and presented as fold changes. M, protein marker in kilodaltons (kDa). The results are expressed as mean ± SEM. * p < 0.05, *** p < 0.001. The results were obtained from at least three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Mutagenesis, Expressing, Transfection, Microscopy, Western Blot, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: SIP increases autophagy in cells that express N-terminal mutant HTT but not in the presence of N-terminal wt HTT. a HEK293T cells were co-transfected with plasmids (control pEntry and e1HTT-25Q-RFP; wt SIP and e1HTT-25Q-RFP; control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP), pretreated with rapamycin and chloroquine, and imaged for autophagy using the Cyto-ID Autophagy detection kit. Signals from autophagosomes were detected in green. Signals that corresponded to the expression of proteins that were encoded by e1HTT-25Q-RFP and e1HTT-72Q-RFP were detected in red and yellow, respectively. Aggregates of e1HTT-72Q were imaged in yellow (white arrows) because of their high fluorescence intensity, which was detected in the FITC channel. Scale bar = 1 µM. b Area of autophagosomes in double-transfected HEK293T cells using the above plasmids. The area of autophagosomes was measured as the number of pixels per e1HTT expressed cell

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Mutagenesis, Transfection, Expressing, Fluorescence

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Ubiquitination of N-terminal wt HTT and mHTT increases in the presence of SIP. Immunoprecipitations (IP) of ubiquitinated exon 1 wt HTT a or mHTT b in the presence of SIP detected by Western blot (WB). a Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-25Q-RFP plasmids or both wt SIP and e1HTT-25Q-RFP plasmids. b Immunoprecipitation was performed in extracts from cells that were co-transfected with both control pEntry and e1HTT-72Q-RFP plasmids or both wt SIP and e1HTT-72Q-RFP plasmids. In a and b , IP was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 HTT ubiquitination (e1HTT-25Q-Ub and e1HTT-25Q-polyUb or e1HTT-72Q-Ub and e1HTT-72Q-polyUb, respectively) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). In b , control IP presents the co-precipitation of exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody. # In the control IP experiments in panel a , bands at 55 kDa that corresponded to exon 1 of wt HTT fused with RFP tag (encoded by e1HTT-25Q-RFP) and IgG heavy chain could not be separated in 8% polyacrylamide gels. Ab, corresponding antibody subjected to IP; pEntry, control pEntry plasmid; SIP wt, wildtype SIP; Total, total protein extract; ►, co-precipitating exon 1 of wt HTT or mHTT or its ubiquitinated forms; IgG, heavy chain of the antibody; M, protein marker in kilodaltons (kDa). As controls in a , total protein extracts were analyzed by WB for the electrophoretic mobility of exon 1 wt and mHTT detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody and the loading control GAPDH was detected by anti-GAPDH antibody. c Level of ubiquitination in total protein extracts from HEK293T cells that were treated with different concentrations of the proteasome inhibitor MG132. d Densitometry of exon 1 mHTT ubiquitination (e1HTT-72Q-Ubiquitin) bands detected in the presence of wt SIP and control pLenti plasmid was calibrated based on the intensity of immunoprecipitated exon 1 mHTT (e1HTT-72Q) and presented as fold changes. Note that despite equal amounts of GAPDH in the total cell extracts there is less mHTT and less mHTT binds to the resin in the IP in the presence of wt SIP compared to control conditions. Therefore, the ubiquitination results from IP were normalized to the amount of HTT that bound to the resin. The results are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. ns, not significant. The results were obtained from at least three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Mutations in the N-terminal domain of SIP decrease the ubiquitination of exon 1 mHTT. a Immunoprecipitation (IP) of ubiquitinated exon 1 mHTT in the presence of SIP and its dimerization mutants detected by Western blot (WB). Immunoprecipitation was performed in extracts from cells that were co-transfected with pEntry and e1HTT-72Q-RFP plasmids, wt SIP and e1HTT-72Q-RFP plasmids, SIP K21W and e1HTT-72Q-RFP plasmids, or SIP T30R_S33E and e1HTT-72Q-RFP plasmids. Immunoprecipitation was performed using anti-HTT N-terminal antibody (HTT) and analyzed for the presence of exon 1 mHTT ubiquitination (e1HTT-72Q-Ub or e1HTT-72Q-polyUb) with the application of an antibody that recognized ubiquitin (Ub; clone P4D1). The detection of co-precipitated exon 1 mHTT (e1HTT-72Q) using anti-HTT N-terminal antibody is shown in the control IP. Ab, corresponding antibody subjected to immunoprecipitation; pE, control pEntry plasmid; SIP wt, wildtype SIP; SIP K21W, mutant SIP K21W; SIP T30R_S33E, mutant SIP T30R_S33E; Total, total protein extract; ►, co-precipitating exon 1 mHTT or its ubiquitinated forms. IgG, heavy chain of antibody; M, protein marker in kilodaltons (kDa). As controls in panel a , total protein extracts were analyzed by WB for the electrophoretic mobility of e1HTT-72Q protein that was detected by N-terminal HTT antibody. SIP was detected by anti-SIP antibody. The loading control, GAPDH, was detected by anti-GAPDH antibody. b Densitometry of exon 1 mHTT ubiquitination bands in the presence of SIP and its dimerization mutants, detected by IP was calibrated based on the intensity of immunoprecipitated e1HTT-72Q in control IP and presented as fold changes. b Note that despite equal amounts of GAPDH in the total cell extracts there is less mHTT and less mHTT binds to the resin in the IP in the presence of wt SIP compared to control conditions and mHTT levels are elevated in the presence of SIP mutants. Therefore, the ubiquitination results from IP were normalized to the amount of mHTT that bound to the resin. The results are expressed as mean ± SEM. * p < 0.05, *** p < 0.001. ns, not significant. The results were obtained from five independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Marker

Journal: Cell & Bioscience

Article Title: Siah-1-interacting protein regulates mutated huntingtin protein aggregation in Huntington’s disease models

doi: 10.1186/s13578-022-00755-0

Figure Lengend Snippet: Mutations in the N-terminal domain of SIP decrease its ability to protect mHTT against aggregation. a HEK293T cells were co-transfected with plasmids (control pEntry and e1HTT-72Q-RFP; wt SIP and e1HTT-72Q-RFP; SIP K21W and e1HTT-72Q-RFP; SIP T30R_S33E and e1HTT-72Q-RFP) and imaged under a fluorescent microscope (left part of panel a ). Images in the right part of the panel a were taken in transmitted light. The size and number of mHTT aggregates (e1HTT-72Q) are presented in panels b and c , respectively. b Red color signals of e1HTT-72Q were measured as the number of pixels per aggregate. Panel b shows the average aggregate size measured for each of the analyzed images. The results are expressed as mean ± SEM. Scale bar = 1 µM. ** p < 0.01; *** p < 0.001. ns, not significant. The results were obtained from three independent HEK293T culture preparations

Article Snippet: The expression of e1HTT-72Q aggregates and e1HTT-25Q protein in HEK293T cells that were transiently co-transfected with e1HTT-72Q-RFP or e1HTT-25Q-RFP plasmids and wt SIP or its dimerization mutants in pCMV-pEntry plasmid or pCMV-pEntry as a control were imaged using an IX70 Microscope (Olympus).

Techniques: Transfection, Microscopy

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: The DENV2 NS2B/NS3 SLC assays. (A) The DENV2 NS2B/NS3 SLC constructs. NLuc aa 1-416-NS2B (named as NLuc-NS2B), NS2B-NLuc aa 1-416 (NS2B-NLuc), NLuc416-NS2B aa 49-66 (NLuc-E66stop), NS2B (aa 49-66)-NLuc416 (E66stop-NLuc), GST-NS3-CLuc aa 398-550 (GNC), and GST-CLuc (aa 398-550)-NS3 (GCN). (B) The positions of NLuc and CLuc are important. Equal concentrations (100 nM) of each pair of NS2B/NS3 constructs were mixed (or alone) and incubated with luciferin substrate. ***P < 0.001. In all bar graphs, means and SD from triplicate experimental data were shown, unless otherwise specified. (C) Effects of detergent at 0.05% concentration. **P < 0.01; ***P < 0.001. (D) Dose-response of NLuc-E66stop/GCN pair. Nluc-E66stop at 20 nM was included in each experiment. Concentration of GCN was varied as indicated. ***P < 0.001. (E) The DENV2 MBP-NS3 fusion protein specifically inhibited the SLC by NLuc-E66stop and GCN (80 nM each). MBP-NS3 or MBP were at 3.25 μM each. ***P < 0.001. (F) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by “cold” MBP-NS3. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (G) NS2B mutations greatly reduced SLC. GCN was paired with equal molar of NLuc-E66stop or NLuc-E66stop mutants (L51A, L53A, and V59A). ***P < 0.001.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Construct, Incubation, Concentration Assay, Inhibition, Software

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: HTS assay identified potent orthosteric protease inhibitors. (A) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by SK-12. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (B) HTS parameters using purified NLuc-E66stop and GCN. NLuc-E66stop and GCN at 100 nM were used with DMSO or SK-12 (40 μM). n = 8. ***P < 0.001. (C) NS3 pockets were accessible to small molecule inhibitor when NS2B and NS3 were co-expressed. Plasmids of NLuc-E66stop and GCN were co-transformed into Escherichia coli BL21 (DE3). Cells were grown to OD600 of 0.6 and were induced by IPTG. Cells were continuously grown for 2 h, collected and resuspended in luciferase assay buffer. About 100 μl of cells was dispensed into a 96-well plate, incubated with 1% DMSO or SK-12 (40 μM) for 2 h, then mixed with substrate luciferin n = 8. ***P < 0.001. (D) Summary of HT screening of the NCGC Pharmaceutical Collection in all plates. Statistics were generated by averaging those of 20 plates that were calculated from 32 wells in each plate. (E) SDS-PAGE analysis of purified His-MBP-NS3 (lane 2) and His-NS2B (Lane 3). Lane 1, Bio-Rad broad range molecular weight (MW) standard. (F) CD spectrum of purified His-MBP-NS3. (G) Sigmoidal curve fittings of dose-response inhibitions of the His-NS2B/His-MBP-NS3 protease activities by drugs. Inset: schematic representations of identified drugs.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: HTS Assay, Inhibition, Software, Purification, Transformation Assay, Luciferase, Incubation, Generated, SDS Page, Molecular Weight

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs directly bind to the NS3 protease domain and disrupt interactions between NS2B and NS3. (A) GST pull-down assay. GST-NS3 or the GST-tag (10 μg) was immobilized on the Glutathione sepharose-4B affinity beads (GE HealthCare). The FLAG-tagged NS2B (10 μg) was incubated with the beads for 2 h, and subjected to western blots (WB), using anti-FLAG (Genscript) and anti-GST antibodies (GE HealthCare). (B) Dose-dependent inhibition of NS2B-NS3 interactions by drugs, using the GST pull-down assay. The assay was performed the same as in A, except that two-fold dilution series of drugs were incubated with the GST-NS3 beads overnight prior to incubation with the FLAG-NS2B. Bottom panels showed normalized binding of FLAG-tag NS2B to GST-NS3. The binding of NS2B to NS3 in the absence of each drug (DMSO control) was set as 100%. The relative binding of NS2B to NS3 in the presence of each drug was normalized to the DMSO control. n = 3. (C) PTSA for binding of drugs to the MBP-NS3 protein. ΔTm was defined as Tm−drug−TmDMSO. (D) SPR sensorgrams of kinetic data for the binding of drugs to refolded NS3. The refolded His-NS3 was coupled to a ProteOn GLH sensor chip (∼15 000 RU). Each drug with three-fold dilutions was injected. Global fitting of data to a 1:1 binding model is shown in dark black.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Pull Down Assay, Incubation, Western Blot, Inhibition, Binding Assay, FLAG-tag, Injection

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs inhibit viral polyprotein precursor (PP) processing. (A) Lineweaver-Burk plot of kinetics experimental data for inhibition of the His-NS2B/His-MBP-NS3 protease complex by drugs. The DENV2 MBP-NS3 (100 nM) was mixed with temoporfin (3, 1.5, and 0.75 μM), niclosamide (30, 15, and 7.5 μM), or nitazoxanide (30, 15, and 7.5 μM) for 30 min. The DENV2 His-NS2B (1 μM) was added together with the Abz substrate at various concentrations (800-25 μM in two-fold dilutions). (B-D) Western blots (WB) analysis of dose-dependent inhibition of ZIKV NS3 expression by temoporfin (B), niclosamide (C), and nitazoxanide (D) using the GTX133309 ZIKV α-NS3 antibody (GeneTex) (left panel), respectively. The experiment was performed at the 48 h time point. Middle panel, NS3 expression (lower bands) normalized to the GAPDH loading control. Right panel, accumulated PP normalized to the DMSO control. **P < 0.01; ***P < 0.001. (E) MS/MS spectra obtained from the fragmentation of the precursor ion at m/z corresponding to representative ZIKV peptides. Fragment ions corresponding to y- and b-ions were observed (red lines).

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Inhibition, Western Blot, Expressing, Tandem Mass Spectroscopy