Figure 1.
scFv-C4-PEST enhances degradation of GFP-tagged httex1 soluble and insoluble protein fragments.
ST14A cells were co-transfected with GFP-tagged httex1-25Q (httex1-25Q-GFP) or (httex1-72Q-GFP), plus either empty vector, scFv-C4, or scFv-C4-PEST plasmids. A. Representative live cell imaging depicting reduction of httex1-72Q-GFP and httex1-25Q-GFP fluorescence in the scFv-C4-PEST co-transfected groups. Phase contrast confirms uniform cell integrity. 48 h; bar = 20 µm. B. SDS-PAGE Western blot and densitometry probed for htt (EM48), quantified vs. actin. Monomeric soluble mhttex1 protein fragments were quantitatively reduced in scFv-C4-PEST vs. scFv-C4 co-transfected cells. (Mean ± SEM; *p<0.05 comparing httex1-25Q-GFP co-transfections; *p<0.05 comparing httex1-72Q-GFP co-transfections) Note that B shows only soluble httex1-72Q fragment levels, which are low unless intrabody is present. C. Agarose Gel Electrophoresis for Resolving Aggregates (AGERA) shows the decrease of detergent-insoluble material in httex1-72Q-GFP to scFv-C4-PEST co-transfected cells compared to other groups.
Figure 2.
scFv-C4-PEST further enhances viability in ST14A co-transfection experiments compared to scFv-C4.
Viability and GFP geometric mean fluorescent intensity (MFI) were assayed by flow cytometric analysis 48 h after transfection. For scFv-C4-PEST vs.scFv-C4: A. The percentage of propidium iodide (PI) positive nonviable cells is reduced. B. Total httex1-25Q-GFP and httex1-72Q-GFP reduction was confirmed. (means ± SEM; *** p<0.001 within groups.)
Table 1.
Physico-chemical properties of scFv-C4 intrabodies.
Figure 3.
Scrambling the PEST sequence and specific inhibition of the proteasome eliminates the enhanced degradation of httex1-72Q-GFP.
A. Representative live cell images of dual transfections. Diffuse httex1-72Q-GFP fluorescence is similar between scFv-C4-PEST-SCR and scFv-C4 cells, and much higher than scFv-C4-PEST. B. Biochemical fractionation of HA-tagged fluorobodies from ST14A cells. Detergent-soluble and -insoluble cell lysates were prepared as described in Materials and Methods. Empty vector and scFv-C4-PEST transfected cells express minimal levels of monomeric htt, while scFv-C4-PEST-SCR and scFv-C4 transfected cells have similar levels of monmeric htt C. AGERA. There is an increase of detergent-insoluble material in scFv-C4-PEST-SCR transfected cells compared to scFv-C4-PEST. D. Western blotting of total protein. Monomeric mhtt levels do not differ between scFv-C4 and scFv-C4-PEST-SCR co-transfected cells. C4-PEST reduces soluble mhtt by ∼90%, and empty vector control mhtt is insoluble E. Proteasome inhibition reduced clearance of insoluble httex1-72Q by scFv-C4-PEST. ST14A cells were co-transfected with httex1-72Q-GFP and scFv-C4-PEST. 36 h after transfection, cells were treated with 10 µM epoxomicin (+), a potent and selective proteasome inhibitor [34]; or DMSO vehicle control. Insoluble mhtt was assessed at 48 h using AGERA. Proteasome inhibition resulted in reduced clearance of htt exon1 protein fragments by scFv-C4-PEST.
Figure 4.
PEST fusion reduces steady-state scFv-C4 intrabody levels modestly, and independent of antigen.
Using dual transfections as above, scFv-HA levels were compared among scFv-C4, scFv-C4-PEST, and scFv-C4-PEST-SCR, in the presence and absence of httex1-72Q-GFP. Levels of scFv-C4-PEST were reduced by ∼25% when compared with scFv-C4 or the scrambled (inactive) PEST. The presence or absence of antigen had no significant effect on intrabody-PEST levels. (n = 4; mean ± SEM; *p<0.05. for C4 vs. C4-PEST/C4-PEST-SCR.)
Figure 5.
Fusion of PEST motif to anti htt fibril-specific scFv-6E intrabody does not degrade htt fragments in ST14A cells.
ST14A cells co-transfected with httex1-72Q-GFP and either empty vector, scFv-6E, or scFv-6E-PEST plasmids. Cells imaged and processed at 48 h. A. Live cell imaging. scFv-6E-PEST does not appear to alter the aggregation of htt compared to empty vector or scFv-6E. B. Western blotting. Monomeric htt is similar between groups. HA-tagged intrabody levels are similar between groups. C. Detergent-insoluble material resolved by AGERA is similar between empty vector and 6E-PEST groups.
Figure 6.
Targeted degradation of htt occurs in a dose-dependent manner.
ST14A cells co-transfected with 1∶2 or 1∶5 transfection ratio of intrabody-PEST to httex1-72Q-GFP plasmids. A. Live cell imaging. Httex1-72Q is diffuse at 1∶2, and being begins to form aggregates at a 1∶5 transfection ratio of intrabody to httex1-72Q-GFP in scFv-C4 transfected cells. Fusion scFv-C4-PEST clears mhtt at 1∶2, and keeps mhtt diffuse at 1∶5. B, C. Western blots of EM48 immunoreactivity measuring the soluble (B; SDS-PAGE) and insoluble (C; AGERA) material to confirm live cell imaging.