Fig 1: Partial inhibition of P4HB alters PrPC glycosylation and decreases PrPC levels in CAD5 cells.To determine whether inhibition of the P4HB a domain affects PrPC homeostasis, uninfected cells were treated with the P4HB a domain-specific inhibitor KSC-34. (A) Schematic structure of P4HB, which features catalytic a and a′ domains (orange), non-catalytic ligand-binding domains b and b′ (green), and an ER-retention motif (KDEL) at the C-terminus. The selective P4HB inhibitor, KSC-34, is 30-fold more selective for the a domain than the a′ domain with an IC50 of ~3.5 μM. (B) Lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h were labeled with or without PEG-PCMal, and then PEG-PCMal labeled proteins were examined by immunoblotting. The blots were probed with antibodies to P4HB, PDIA3, and PDIA4. (C) Representative immunoblots for PrPC, P4HB, GRP78, PDIA3, PDIA4, and actin in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. (D, E) Quantification of PrPC and P4HB (D) as well as GRP78, PDIA3, and PDIA4 (E) levels in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). (F) Quantification of PrPC glycoforms in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). (G) Immunofluorescence images of CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h. The expression of PrPC (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). (H) Representative immunoblots of PrPC, P4HB, GRP78, and actin in cell lysates from CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34. (I) Quantification of PrPC levels in CAD5 cells treated for 1, 2, or 3 days with 0 or 3.5 µM KSC-34 (n = 4). (J) Representative immunoblots for detergent-insoluble PrP and P4HB species in cell lysates from CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. (K) Quantification of the ratio of insoluble:total PrP in CAD5 cells treated with 0 or 3.5 µM KSC-34 for 72 h (n = 4). (L) Quantification of detergent-insoluble P4HB levels in CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h (n = 4). All data are mean ± SEM. In panels C, H, and J, PrP was detected using the antibodies D18 or POM1. In panels D, E, K, and L, statistical significance was assessed using unpaired two-tailed t tests. In panels F and I, statistical significance was assessed using a two-way ANOVA followed by Šídák’s multiple comparison test.
Fig 2: P4HB increases disulfide bond formation efficiency in PrPC.To evaluate whether P4HB directly affects disulfide bond formation in PrPC, an in vitro assay that assesses disulfide status in recombinant PrP was conducted in the presence or absence of recombinant P4HB. (A) Schematic of PEG-Maleimide (PEG-Mal) assay for assaying disulfide bond formation in recombinant mouse PrP (recMoPrP). (B) recMoPrP was reduced with tris(2-carboxyethyl)phosphine (TCEP), treated with (+) or without (-) recombinant P4HB (recP4HB), and then proteins were labeled with PEG-Mal. PEG-Mal labeled proteins were resolved by SDS-PAGE and analyzed by immunoblotting. The blot was probed with the anti-PrP antibody SAF32 or an anti-P4HB antibody. (C) Quantification of the relative ratio of PEG-Mal-labeled to unlabeled PrP for reactions performed in the absence (-) and presence (+) of recP4HB. Data are mean ± SEM from four independent replicates. Statistical significance was assessed using an unpaired, two-tailed t test.
Fig 3: Transient transfection with a secreted version of P4HB increases PrPres levels in CAD5 cells.To assess whether overexpression of P4HB modulates prion replication, cells were transiently transfected with constructs encoding WT or secreted P4HB variants. (A) Immunoblots for PrPC, P4HB, and actin in total cell lysates as well as unbound and eluate fractions following incubation with streptavidin agarose from CAD5 cells subjected to cell surface biotinylation (+) or mock (-) treatment indicate that a proportion of P4HB is present at the cell surface. (B) Schematic structures of WT mouse P4HB as well as mutants with a catalytically inactive a domain (C55/58S) and/or deletion of the C-terminal ER retention signal (ΔKDEL). (C) Immunoblot of PrPC, P4HB, and actin levels in cell lysates from CAD5 cells transiently transfected with empty vector (EV) or with WT or mutant P4HB for 48 h. (D) Quantification of PrPC and P4HB levels in lysates from transiently transfected CAD5 cells (n = 4 independent replicates). (E) Immunoblot of secreted PrP and P4HB levels in the conditioned medium from CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. As a control, the membrane was stained for total protein following transfer using Ponceau S. (F) Representative immunoblots for proteinase K (PK)-resistant PrPSc (PrPres) levels in lysates from either RML or 22L prion-infected CAD5 cells transiently transfected with EV or with WT or mutant P4HB for 48 h. (G) Quantification of PrPres levels in transiently transfected RML or 22L prion-infected CAD5 cells (n = 4 independent replicates). In panels A, C, and E, PrP was detected using the antibody D18 whereas in panel F, PrPres was detected using the antibody D13. In panels D and G, graphs display mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.
Fig 4: Inhibition of the P4HB a domain increases PrPres levels in prion-infected CAD5 cells.To determine whether inhibition of the P4HB a domain affects prion replication, prion-infected cells were treated with the P4HB inhibitor KSC-34 and then PrPres levels were measured. (A) Representative immunoblots for total PrP, P4HB and proteinase K (PK)-resistant PrPSc (PrPres) levels in lysates from either RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 for 72 h. The blots were probed with the anti-PrP antibody D13, an anti-P4HB antibody, and an anti-actin antibody. (B-D) Quantification of total PrP (B), PrPres (C), and P4HB (D) levels in RML or 22L prion-infected CAD5 cells treated with 0 or 3.5 μM KSC-34 (n = 4 independent replicates). (E) Representative immunoblot of PrPres levels in RML prion-infected CAD5 cells treated with 0, 3.5, or 7 µM KSC-34 for 72 h. (F) Quantification of PrPres levels in RML prion-infected CAD5 cells treated with 0, or 3.5, or 7 µM KSC-34 (n = 4 independent replicates). (G) Representative immunoblot of PrPres levels in CAD5 cells challenged with RML prions in the presence or absence of KSC-34 and then passaged 4 times in the continuous presence of either 0 or 3.5 µM KSC-34. (H) Quantification of PrPres levels in CAD5 cells challenged with RML prions in the presence or absence of KSC-34 and then treated with 0 or 3.5 µM KSC-34 for 4 passages (n = 6 independent replicates). In panels B-D, F, and H, graphs display mean ± SEM. In panels A, E, and G, PrP and PrPres were detected using the antibody D13. Statistical significance in panels B-D, and H was assessed using unpaired, two-tailed t tests. In panel F, statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.
Fig 5: Stable knock-down of P4HB decreases PrPC levels and hinders de novo prion infection.To determine whether P4HB modulates prion infection, stable shRNA-mediated P4HB knock-down cell lines were challenged with two different prion strains and then PrPres levels were measured. (A) Representative immunoblots for PrPC, P4HB, GRP78, PDIA3, PDIA4, and actin in cell lysates from CAD5 cells stably transfected with control or one of two distinct P4HB shRNAs. (B, C) Quantification of PrPC and P4HB levels (B) as well as GRP78, PDIA3, and PDIA4 levels (C) in CAD5 cells stably transfected with control or P4HB shRNA (n = 5). (D) Immunofluorescence images of CAD5 cells stably transfected with control or P4HB shRNA. PrPC (green) and P4HB (red) were revealed using the anti-PrP antibody POM1 and an anti-P4HB antibody, and nuclei were stained with DAPI (blue). Scale bar = 10 μm (applies to all images). (E) Immunoblots of detergent-insoluble PrP and P4HB species in CAD5 cells stably transfected with control or P4HB shRNA. (F) Quantification of the ratio of insoluble:total PrP in CAD5 cells stably transfected with control or P4HB shRNA (n = 4). (G, I) Immunoblots of proteinase K (PK)-resistant PrP (PrPres) levels in CAD5 cells stably transfected with control or P4HB shRNA and then infected with either RML (G) or 22L (I) prions. (H, J) Quantification of PrPres levels in CAD5 cells stably transfected with control or P4HB shRNA and then infected with either RML (H) or 22L (J) prions (n = 8). In panels A and E, PrP was detected using the antibody D18 whereas in panels G and I, PrPres was detected using the antibody D13. In panels B, C, F, H, and J, data are mean ± SEM and statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.
Supplier Page from Sino Biological, Inc. for Mouse P4HB Gene ORF cDNA clone expression plasmid