ubxd8  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Article Snippet: Caspase-4 (Cat# 4450S, 1:1000), Gasdermin D (Cat# 39754S, 1:1000), Cleaved Gasdermin D (Asp275) (Cat# 36425S, 1:500), Cleaved Gasdermin D (Asp276) (Cat# 10137S, 1:1000), NLRP3 (Cat# 15101S, 1:1000), Tubulin (Cat# 2144S, 1:2000), Actin (Cat#4967S, 1:2000), UBXN3B (Cat# 34945S, 1:1000), K63-linkage specific polyubiquitin (Clone D7A11, Cat# 5621S,1:500), K48-linkage specific polyubiquitin (Clone D9D5, Cat# 12805S, 1:1000), Phospho-Jak1 (Tyr1034/1035) (Cat# 74129S, 1:1000), Phospho-Stat1 (Tyr701) (Cat# 9167S, 1:1000), Myc-Tag (Cat# 2276S, 1:2000) and HA-Tag (Clone C29F4, Cat# 3724S, 1:1000) antibodies were from Cell Signaling.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Knock-Out, Mutagenesis

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Volcano plot of the (−log10-transformed P value versus the log2-transformed ratio of wildtype/ UBXD8 KO) proteins identified from HeLa cells. n = 3 biologically independent samples for each genotype. P values were determined by empirical Bayesian statistical methods (two-tailed t test adjusted for multiple comparisons using Benjamini-Hochberg’s correction method) using the LIMMA R package; for parameters, individual P values and q values, see Supplementary Dataset. Peroxisomal proteins important for biogenesis (dark blue) and metabolism (red) are highlighted. This dataset has been previously published in and is reanalyzed here. B. Schematic of tandem mass tag (TMT) proteomic hits in distinct peroxisomal pathways. C. Peroxisomal proteins identifies in (A) show reduced expression in UBXD8 KO compared to wildtype HeLa and Hek293T cells. D. Quantification of (C). **** : P<0.0001, N=3, Unpaired T-test.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transformation Assay, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. HeLa wildtype and UBXD8 KO cells stained for peroxisomes using peroxisomal matrix marker catalase B. Quantification of average peroxisome per cell and average peroxisome size from (A). At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals **** : P<0.0001, N=3, Unpaired T-test. C. Peroxisome abundance in wildtype and UBXD8 KO cells that have either no peroxisomes or less then 10 peroxisomes per cell. D. Rescue of peroxisome number in UBXD8 KO cells transfected with either UBXD8-HA, UBXD8-UBA*-HA, UBXD8-ΛUAS-HA or UBXD8-UBX*-HA. Cells were stained for peroxisomes using peroxisomal matrix marker catalase. E. Quantification of average peroxisome per cell from HeLa wildtype and UBXD8 KO cells as well as UBXD8 KO cells transfected with either UBXD8-HA, UBXD8-UBA*-HA, UBXD8-ΛUAS-HA or UBXD8-UBX*-HA (D). Peroxisome numbers were quantified in cells expressing HA-tagged UBXD8 constructs only. At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals **, **** : P<0.05, 0.0001. Two-way ANOVA with Tukey’s multiple comparisons test. F. Immunoblots of constructs transfected. Scale bar is 10μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Staining, Marker, Transfection, Expressing, Construct, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Hela cells were transfected with siRNAs to HRD1, and cells were stained for catalase (see also Supplementary Figure 3). Quantification of number of peroxisomes per cell, at least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. ns: not significant, **** : P<0.0001. One-way ANOVA with Dunnett’s multiple comparisons test. B. Quantification of peroxisome per cell in HEK293T wildtype and GP78 KO cells. At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** : P<0.0001. Unpaired T-test. C. HeLa wildtype and UBXD2 KO cells stained for peroxisomes using catalase. D. Quantification of peroxisome per cell from (C). At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. NS: not significant. Unpaired T-test. E. Immunoblots of Hela wildtype (untreated or treated with DTT) and UBXD8 KO for ER stress markers BiP and ATF4. N=3, F. rt-qPCR of xbp1 total and xbp1 spliced (xbp1s) mRNA transcripts in wildtype and UBXD8 KO cells treated with 1.5 mM DTT for 4 hours. N=3. NS: Not significant, *, **** : P < 0.01, 0.0001. Two-way ANOVA with Dunnett’s post-hoc analysis. Scale bar is 10μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Staining, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Volcano plot of the total cholesterol esters and triacylglycerol species identified using lipidomics of whole cell extracts of HEK-293T cells (−log10-transformed P value versus the log2-transformed ratio of UBXD8 KO: wildtype). VLCFA species indicated for CE (orange) and TG (dark blue). This dataset has been previously published in and is reanalyzed here. B. VLCFA species indicated for phosphatidylserine (PS) (green), phosphatidylethanolamine (PE) (red) and phosphatidylcholine (PC) (violet). Lipids were measured by LC-MS/MS following normalization by total protein amount. (n ≥ 3 biologically independent experiments were performed, each with duplicate samples). This dataset has been previously published in and is reanalyzed here. C. Immunoblots of catalase levels in whole cell lysates of HeLa wildtype and UBXD8 KO cells. D. Quantification of catalase levels in (C). N=3 independent experiments. NS: not significant. Unpaired T-test. E. Catalase activity was quantified using a commercial kit. N=3 independent experiments. ** : P<0.0001, Unpaired T-Test.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. GFP-UBXD8 and RFP-SKL were transiently transfected into COS-7 or HeLa cells and stained with BODIPY (665/676) to label lipid droplets. B. HeLa cells were transfected with FLAG-tagged wildtype UBXD8 or UBXD8 domain deletions (UBA, UAS and UBX) (in green) and RFP-SKL (in red). C . Quantification of (B) showing number of peroxisomes with UBXD8 localization. 15-20 cells were analyzed in N=3 independent experiments. Scatter plot shows mean and std.dev. ns: not significant, ** : P<0.001. One-way ANOVA with Dunnett’s multiple comparisons test. Scale bar is 5μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Staining

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Schematic for pexophagy flux reporter. B. Representative images of wildtype and UBXD8 KO cells transfected with GFP-Cherry-PEX26. C. Wildtype and UBXD8 KO cells were transfected with the flux reporter and treated with 150 nM Torin1 for 18 hours. Quantification showing the ratio of GFP: (GFP + mCherry + ) in Hela wildtype and UBXD8 KO cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **, ***, **** : P<0.01, 0.001, 0.0001. Two-way ANOVA with Šidáks multiple comparisons test. D. Representative images of HeLa control or p97 siRNAs and GFP-Cherry-PEX26. E. Quantification showing the ratio of GFP: (GFP + mCherry + ) in Hela control, or p97 depleted cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. *, **, **** : P<0.05, 0.001, 0.0001. Two-way ANOVA with Tukey’s multiple comparisons test. F. Immunoblot showing p97. Scale bar is 10 μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Representative images of HeLa cells transfected with control and UBXD8 or ATG5 siRNAs and stained for catalase. B. Quantification of peroxisomes per cell from (A). 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** = P<0.0001. Two-way ANOVA with Dunnett’s multiple comparisons test. C. Immunoblot of UBXD8 and ATG5. D. Representative images of HeLa cells (wildtype and UBXD8 KO) transfected with GFP-USP30. Cells were stained for catalase. E. Quantification of peroxisomes per cell in GFP-USP30 transfected cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. ***, **** = P< 0.001, 0.0001. Two-way ANOVA with Dunnett’s multiple comparisons test N=3, 2-way ANOVA. F. Immunoblot of GFP-USP30 expression. G. Wildtype and UBXD8 KO HeLa cells were stained with NBR1 and catalase. H. Mander’s colocalization of images in (G). 100-120 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** :P< 0.0001. Students unpaired T-test. F. Immunoblot of GFP-USP30 expression. Scale bar is 10 μM (A and D) and 5 μM (G).

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Control, Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. HEK293T cells were treated with 150 nM Torin or 50 nM bafilomycin A (BafA) for 24 hours and then with 100 μg/ ml cycloheximide for the indicated time points to stop translation. Immunoblots showing PMP70 half-life. B. Quantification of PMP70 levels normalized to GAPDH from (A). N=3 independent experiments. **, *** : P< 0.01, 0.001. Two-way ANOVA with Tukey’s multiple comparisons test. C. HEK293T cells were transfected with HA-ubiquitin and treated with 150 nM Torin-1 in the presence or absence of 5 μM Bortezomib (Btz) or 50 nM BafA for 24 hours. Cells were lysed in SDS, and denaturing HA immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PMP70 in cells treated with Btz or BafA. N=3 independent experiments. D. HEK293T cells were transfected with HA-ubiquitin and siRNAs to control, UBXD8 or p97. Cells were treated with 150 nM Torin-1 for 24 hours. Cells were lysed in SDS, and denaturing HA immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PMP70 in cells depleted of UBXD8 and p97. N=3 independent experiments. E. Model showing p97-UBXD8 suppression of pexophagy.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Western Blot, Transfection, Control

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Volcano plot of the (−log10-transformed P value versus the log2-transformed ratio of wildtype/ UBXD8 KO) proteins identified from HeLa cells. n = 3 biologically independent samples for each genotype. P values were determined by empirical Bayesian statistical methods (two-tailed t test adjusted for multiple comparisons using Benjamini-Hochberg’s correction method) using the LIMMA R package; for parameters, individual P values and q values, see Supplementary Dataset. Peroxisomal proteins important for biogenesis (dark blue) and metabolism (red) are highlighted. This dataset has been previously published in and is reanalyzed here. B. Schematic of tandem mass tag (TMT) proteomic hits in distinct peroxisomal pathways. C. Peroxisomal proteins identifies in (A) show reduced expression in UBXD8 KO compared to wildtype HeLa and Hek293T cells. D. Quantification of (C). **** : P<0.0001, N=3, Unpaired T-test.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transformation Assay, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. HeLa wildtype and UBXD8 KO cells stained for peroxisomes using peroxisomal matrix marker catalase B. Quantification of average peroxisome per cell and average peroxisome size from (A). At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals **** : P<0.0001, N=3, Unpaired T-test. C. Peroxisome abundance in wildtype and UBXD8 KO cells that have either no peroxisomes or less then 10 peroxisomes per cell. D. Rescue of peroxisome number in UBXD8 KO cells transfected with either UBXD8-HA, UBXD8-UBA*-HA, UBXD8-ΛUAS-HA or UBXD8-UBX*-HA. Cells were stained for peroxisomes using peroxisomal matrix marker catalase. E. Quantification of average peroxisome per cell from HeLa wildtype and UBXD8 KO cells as well as UBXD8 KO cells transfected with either UBXD8-HA, UBXD8-UBA*-HA, UBXD8-ΛUAS-HA or UBXD8-UBX*-HA (D). Peroxisome numbers were quantified in cells expressing HA-tagged UBXD8 constructs only. At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals **, **** : P<0.05, 0.0001. Two-way ANOVA with Tukey’s multiple comparisons test. F. Immunoblots of constructs transfected. Scale bar is 10μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Staining, Marker, Transfection, Expressing, Construct, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Hela cells were transfected with siRNAs to HRD1, and cells were stained for catalase (see also Supplementary Figure 3). Quantification of number of peroxisomes per cell, at least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. ns: not significant, **** : P<0.0001. One-way ANOVA with Dunnett’s multiple comparisons test. B. Quantification of peroxisome per cell in HEK293T wildtype and GP78 KO cells. At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** : P<0.0001. Unpaired T-test. C. HeLa wildtype and UBXD2 KO cells stained for peroxisomes using catalase. D. Quantification of peroxisome per cell from (C). At least 100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. NS: not significant. Unpaired T-test. E. Immunoblots of Hela wildtype (untreated or treated with DTT) and UBXD8 KO for ER stress markers BiP and ATF4. N=3, F. rt-qPCR of xbp1 total and xbp1 spliced (xbp1s) mRNA transcripts in wildtype and UBXD8 KO cells treated with 1.5 mM DTT for 4 hours. N=3. NS: Not significant, *, **** : P < 0.01, 0.0001. Two-way ANOVA with Dunnett’s post-hoc analysis. Scale bar is 10μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Staining, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Volcano plot of the total cholesterol esters and triacylglycerol species identified using lipidomics of whole cell extracts of HEK-293T cells (−log10-transformed P value versus the log2-transformed ratio of UBXD8 KO: wildtype). VLCFA species indicated for CE (orange) and TG (dark blue). This dataset has been previously published in and is reanalyzed here. B. VLCFA species indicated for phosphatidylserine (PS) (green), phosphatidylethanolamine (PE) (red) and phosphatidylcholine (PC) (violet). Lipids were measured by LC-MS/MS following normalization by total protein amount. (n ≥ 3 biologically independent experiments were performed, each with duplicate samples). This dataset has been previously published in and is reanalyzed here. C. Immunoblots of catalase levels in whole cell lysates of HeLa wildtype and UBXD8 KO cells. D. Quantification of catalase levels in (C). N=3 independent experiments. NS: not significant. Unpaired T-test. E. Catalase activity was quantified using a commercial kit. N=3 independent experiments. ** : P<0.0001, Unpaired T-Test.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. GFP-UBXD8 and RFP-SKL were transiently transfected into COS-7 or HeLa cells and stained with BODIPY (665/676) to label lipid droplets. B. HeLa cells were transfected with FLAG-tagged wildtype UBXD8 or UBXD8 domain deletions (UBA, UAS and UBX) (in green) and RFP-SKL (in red). C . Quantification of (B) showing number of peroxisomes with UBXD8 localization. 15-20 cells were analyzed in N=3 independent experiments. Scatter plot shows mean and std.dev. ns: not significant, ** : P<0.001. One-way ANOVA with Dunnett’s multiple comparisons test. Scale bar is 5μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Staining

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Schematic for pexophagy flux reporter. B. Representative images of wildtype and UBXD8 KO cells transfected with GFP-Cherry-PEX26. C. Wildtype and UBXD8 KO cells were transfected with the flux reporter and treated with 150 nM Torin1 for 18 hours. Quantification showing the ratio of GFP: (GFP + mCherry + ) in Hela wildtype and UBXD8 KO cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **, ***, **** : P<0.01, 0.001, 0.0001. Two-way ANOVA with Šidáks multiple comparisons test. D. Representative images of HeLa control or p97 siRNAs and GFP-Cherry-PEX26. E. Quantification showing the ratio of GFP: (GFP + mCherry + ) in Hela control, or p97 depleted cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. *, **, **** : P<0.05, 0.001, 0.0001. Two-way ANOVA with Tukey’s multiple comparisons test. F. Immunoblot showing p97. Scale bar is 10 μM.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. Representative images of HeLa cells transfected with control and UBXD8 or ATG5 siRNAs and stained for catalase. B. Quantification of peroxisomes per cell from (A). 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** = P<0.0001. Two-way ANOVA with Dunnett’s multiple comparisons test. C. Immunoblot of UBXD8 and ATG5. D. Representative images of HeLa cells (wildtype and UBXD8 KO) transfected with GFP-USP30. Cells were stained for catalase. E. Quantification of peroxisomes per cell in GFP-USP30 transfected cells. 50-100 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. ***, **** = P< 0.001, 0.0001. Two-way ANOVA with Dunnett’s multiple comparisons test N=3, 2-way ANOVA. F. Immunoblot of GFP-USP30 expression. G. Wildtype and UBXD8 KO HeLa cells were stained with NBR1 and catalase. H. Mander’s colocalization of images in (G). 100-120 cells were analyzed in N=3 independent experiments. Violin plot shows median and 95% confidence intervals. **** :P< 0.0001. Students unpaired T-test. F. Immunoblot of GFP-USP30 expression. Scale bar is 10 μM (A and D) and 5 μM (G).

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Transfection, Control, Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy

doi: 10.1101/2024.09.24.614749

Figure Lengend Snippet: A. HEK293T cells were treated with 150 nM Torin or 50 nM bafilomycin A (BafA) for 24 hours and then with 100 μg/ ml cycloheximide for the indicated time points to stop translation. Immunoblots showing PMP70 half-life. B. Quantification of PMP70 levels normalized to GAPDH from (A). N=3 independent experiments. **, *** : P< 0.01, 0.001. Two-way ANOVA with Tukey’s multiple comparisons test. C. HEK293T cells were transfected with HA-ubiquitin and treated with 150 nM Torin-1 in the presence or absence of 5 μM Bortezomib (Btz) or 50 nM BafA for 24 hours. Cells were lysed in SDS, and denaturing HA immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PMP70 in cells treated with Btz or BafA. N=3 independent experiments. D. HEK293T cells were transfected with HA-ubiquitin and siRNAs to control, UBXD8 or p97. Cells were treated with 150 nM Torin-1 for 24 hours. Cells were lysed in SDS, and denaturing HA immunoprecipitations were performed. Immunoblots of indicated proteins showing increased ubiquitylation of PMP70 in cells depleted of UBXD8 and p97. N=3 independent experiments. E. Model showing p97-UBXD8 suppression of pexophagy.

Article Snippet: CB-5083 was from Selleckchem. siRNAs were purchased from Ambion (Thermo Fisher Scientific): UBXD8-0 (s23260), UBXD8-9 (s23259).

Techniques: Western Blot, Transfection, Control

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Schematic depicting LD biogenesis from the ER membrane. ERTOLD hairpin proteins are considered to integrate first into the cytosolic leaflet of the ER membrane in a monotopic topology, which presumably enables them to partition from the ER bilayer to the LD monolayer membrane during LD biogenesis. NL neutral lipids. b Dual localization of opUBXD8 53-153 mCherry to the ER and LDs. Top: Schematic outline of the opsin (op) and mCherry-tagged opUBXD8 53-153 mCherry construct. HR hydrophobic region. Bottom: Fluorescence micrographs of oleate-treated cells transfected with opUBXD8 53-153 mCherry representative for 3 independent experiments. LipidTox marks LDs. Scale bar: 10 µm. c Isolation of opUBXD8 53-153 mCherry-containing LDs from cells. Left: Schematic outline for isolation of UBXD8-containing LDs. Right: immunoblot of post-nuclear supernatant (PNS), membranes (M), cytosol (C), and LD fractions derived from oleate-treated cells expressing OpUBXD8 53-153 mCherry using anti-calnexin (ER-resident protein), anti-tubulin (cytosolic protein) and anti-mCherry antibodies. Non-transfected cells (NT) serve as specificity control for the antibody. Data are representative for 3 independent experiments. d Integration of opUBXD8 53-153 mCherry into rough microsomes (RMs). Left: Schematic outline of co-translational protein insertion into RMs employing in vitro translation of UBXD8 mRNAs in rabbit reticulocyte lysate (RRL) with subsequent fractionation into soluble and membrane-inserted proteins by centrifugation. Right: Immunoblot of soluble (S) and membrane-inserted (M) fractions derived from in vitro translations reactions using anti-mCherry antibodies (representative for n = 3 independent experiments). mRNA encoding either opUBXD8 53-153 mCherry or UBXD8 53-153 mCherryOP and RMs were added to the reaction as indicated. Arrows indicate glycosylated forms of the respective proteins. e – g opUBXD8 53-153 mCherry single cysteine mutants can be PEGylated in ER bilayer and LD monolayer membranes when the cysteine is solvent-exposed. Top: Principle of solvent-accessibility probing of opUBXD8 53-153 mCherry single cysteine mutants by PEGylation in ER bilayer and LD monolayer membranes, respectively. Only solvent-exposed cysteines are accessible to mPEG forming covalent adducts, while bilayer-embedded cysteines are not reactive with mPEG. Bottom: Proof-of-concept immunoblots probed with anti-mCherry antibodies after PEGylation reaction on RM-inserted and LD-inserted opUBXD8 53-153 mCherry single cysteine mutants as indicated. Non-PEGylated proteins are indicated by (0 PEG) and PEGylated proteins by (1 PEG). TX-100: Triton X-100. Quantifications for multiple replicates of these experiments are shown in Fig. .

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Construct, Fluorescence, Transfection, Isolation, Western Blot, Derivative Assay, Expressing, Control, In Vitro, Fractionation, Centrifugation, Solvent

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Atomistic MD simulations of UBXD8 80-128 in a POPC bilayer membrane with a deeply inserted starting structure. Left: Starting structure Right: average structure after 2 µs. b Atomistic MD simulations of UBXD8 80-128 in a POPC bilayer membrane with a partially inserted starting structure. Left: Starting structure Right: average structure after 2 µs. c Atomistic MD simulations of UBXD8 80-128 in a POPC-triolein/cholesteryl-oleate-POPC trilayer system mimicking the LD monolayer membrane. Left: Starting structure; Middle: side view of average structure after 2 µs; Right: top view of average structure after 2 µs; d Center of mass distances of the Cα atom in P102 of UBXD8 80-128 and the P atom in the phospholipid headgroup (dotted line) during the simulation time of 2 µs, and upon UBXD8 80-128 insertion into POPC bilayers or into trilayer systems as indicated. PI partially inserted, DI deeply inserted, TRIO Triolein, CLOL (cholesteryl-oleate). e Center of mass distances of amino acid Cα atoms in UBXD8 80-128 and the P atom in the phospholipid headgroup (dotted line) in the average structures obtained after 2 µs simulations. UBXD8 80-128 was inserted into POPC bilayers or into trilayer systems as indicated. Max. penetration into bilayer is ~2 nm and into monolayer is ~1 nm. Five independent simulations with the CHARMM36m force field over 2 μs were performed. For ( d ) and ( e ): Lines and shaded areas show mean and ±SEM, respectively ( n = 5 simulations). Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Positioning of the amino acids L91 and L118 within the atomistic starting structure of UBXD8 80-128 that were mutated to a cysteine pair in opUBXD8 53-153 L91C_L118C_mCherry. Cα atoms are highlighted as purple spheres. b Violin plots of distances between the Cα atoms of amino acids L91 and L118 in MD simulations in different membrane systems as shown in Figs. , respectively. DI deeply inserted, TRIO Triolein, CLOL (cholesteryl-oleate). Vertical bars indicate median and maximum/minimum values of the distributions ( n = 5 simulations). c Schematic representation to illustrate the principle of the combined intramolecular crosslinking - PEGylation assay. For clarity, a double-cysteine-containing peptide is only schematically depicted in a bilayer membrane and does not particularly reflect closed or open conformations of UBXD8 in different types of membranes. d , e Immunoblots using anti-mCherry antibodies showing intramolecular crosslinking/PEGylation experiments of opUBXD8 53-153 L91C_L118C_mCherry in either RMs ( d ) or in isolated LDs from cells ( e ). Non-PEGylated protein species (0 PEG) as well as species with one mPEG (1 PEG) or two mPEG (2PEG) molecules attached, are indicated. High molecular weight adducts derived from inter -molecular crosslinking of opUBXD8 53-153 L91C_L118C_mCherry on LDs are indicated (inter x-link). f Quantification of relative opUBXD8 53-153 L91C_L118C_mCherry intramolecular crosslinking efficiencies in RMs versus LDs. From experiments as shown in ( d , e ), bands corresponding to non-PEGylated/intramolecularly crosslinked OpUBXD8 53-153 L91C_L118CmCherry (0 PEG) were quantified. The relative increase in these bands upon addition of crosslinker (lane 2 versus negative DMSO control in lane 1) was calculated from three independent experiments and the values were normalized to the highest value, which was set to 100%. Scatter plots show the mean values with SEM from three independent experiments as well as the individual values for each replicate. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Western Blot, Isolation, High Molecular Weight, Derivative Assay, Control

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a Average atomistic MD simulation structure of UBXD8 80-128 in a POPC bilayer (as in Fig. ) indicating amino acids that were substituted for single cysteines for cwEPR analyses. b Left: Schematic outline of proteo-aLDs generation from proteo-SUVs and isolation by density gradient centrifugation. Right: photograph showing density gradients after centrifugation with floating aLDs when triolein was present during the reconstitution (+). (−): negative control without triolein. c Immunoblot analysis of proteo-aLDs isolation by density gradient fractionation as indicated in ( b ) using anti-S-tag antibodies. Top: negative control without triolein. Arrowhead indicates MTSL-labeled sUBXD8 71-132 His S127C in the top floating aLD fraction when triolein was present during the reconstitution. Representative for n = 3 independent experiments. d Fluorescence micrograph of the top floating aLDs fraction as shown in ( b ) upon reconstitution of Atto488-labeled sUBXD8 71-132 His T130C (green). LipidTox Red marks the neutral lipid core (red). Scale bar: 10 µm. Representative for n = 3 independent experiments. e First derivative absorption cwEPR spectra of MTSL spin-labeled sUBXD8 71-132 -His single cysteine mutants in POPC/DOPS SUVs (left) and aLDs (right). Spectra were normalized by the height of the central EPR line. Asterisks mark spectra with additional shoulders in the low-field region indicating immobile (i) and mobile (m) motional components. f Schematic illustration of how the spin-label positioning in a membrane protein affects the line shape of cwEPR spectra and the membrane depth parameter (Φ). Solvent-exposed: orange; membrane-associated: red; membrane-embedded: blue. In the bilayer midplane, the O 2 concentration is the highest (green), while NiEDDA is gradually excluded from the membrane (purple). g Exemplary EPR power saturation plots of MTSL spin-labeled sUBXD8 71-132 His Y81C and L118C single cysteine mutants reconstituted into SUVs. The peak-to-peak amplitude of the central EPR line was plotted against the square root of the applied microwave power. Power saturation curves were measured under three conditions: nitrogen gas as control (red circles), molecular O 2 (black squares), and NiEDDA (blue diamonds). P 1/2 values were obtained after curve fitting and are indicated with SEM. h Membrane depth parameter (Φ) analysis of MTSL spin-labeled sUBXD8 71-132 His single cysteine mutants in SUVs using cwEPR power saturation analyses ( n = 2 independent experiments). Positive Φ: membrane-embedding; negative Φ: solvent exposure. i Membrane depth parameter (Φ) analysis of MTSL spin-labeled sUBXD8 71-132 His Y96C and I113C reconstituted into either SUVs or aLDs using cwEPR power saturation analyses ( n = 2 independent experiments). Φ close to 0: proximity to solvent-membrane interface. Results for SUVs are duplicates from ( h ) for direct comparison with aLDs. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Isolation, Gradient Centrifugation, Centrifugation, Negative Control, Western Blot, Fractionation, Labeling, Fluorescence, Membrane, Solvent, Concentration Assay, Control, Comparison

Journal: Nature Communications

Article Title: Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements

doi: 10.1038/s41467-024-48843-8

Figure Lengend Snippet: a MARTINI-based coarse-grained simulation of a bilayer-embedded LD lens consisting of triolein/cholesteryl oleate. Upper panel: UBXD8 80-128 was integrated into the bilayer in its shallow conformation as assessed by atomistic simulations. After 5 μs, the peptides have partitioned to the LD monolayer surface where they accumulate. Lower panel: When inserted into the bilayer in the deep-V state as suggested by atomistic simulations, UBXD8 80-128 accumulates at the bilayer-LD rim but does not transition to the LD surface within 5 µs. b , c All-atom pulling simulations reveal high free-energy costs involved in pulling the shallow-open state of UBXD8 80-128 downwards ( b ) or the deep-V state upwards ( c ) within a planar POPC bilayer membrane. d All-atom simulation system of a minimal LD-bilayer system used for pulling the deep-V-inserted UBXD8 80-128 along the LD rim towards the open-shallow conformation. e Graphs comparing free-energy profiles during MD pulling experiments of UBXD8 80-128 in planar bilayers versus at the LD rim as derived from ( b – d ). Shaded areas show SEM derived by bootstrapping from the set of 33 umbrella histograms. f Revised model for the intramembrane positioning of UBXD8 in ER bilayer versus LD monolayer membranes indicating that structural rearrangements are required for enabling the partitioning. Positive charges are indicated by “+”. Source data are provided as a source data file.

Article Snippet: In order to experimentally determine the insertion depth of UBXD8 in bilayer and monolayer membranes, respectively, we established continuous-wave electron paramagnetic resonance (cwEPR) spectroscopy workflows of UBXD8 in different biomimetic model membranes.

Techniques: Membrane, Derivative Assay