scFv-C4-PEST further enhances viability in ST14A co-transfection experiments compared to scFv-C4.

<p>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: <b>A</b>. The percentage of propidium iodide (PI) positive nonviable cells is reduced. <b>B</b>. Total httex1-25Q-GFP and httex1-72Q-GFP reduction was confirmed. (means ± SEM; *** p<0.001 within groups.)</p

Targeted degradation of htt occurs in a dose-dependent manner.

<p>ST14A cells co-transfected with 1∶2 or 1∶5 transfection ratio of intrabody-PEST to httex1-72Q-GFP plasmids. <b>A</b>. 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>B, C</b>. Western blots of EM48 immunoreactivity measuring the soluble (<b>B</b>; SDS-PAGE) and insoluble (<b>C</b>; AGERA) material to confirm live cell imaging.</p

Fusion of PEST motif to anti htt fibril-specific scFv-6E intrabody does not degrade htt fragments in ST14A cells.

<p>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. <b>A</b>. Live cell imaging. scFv-6E-PEST does not appear to alter the aggregation of htt compared to empty vector or scFv-6E. <b>B</b>. Western blotting. Monomeric htt is similar between groups. HA-tagged intrabody levels are similar between groups. <b>C</b>. Detergent-insoluble material resolved by AGERA is similar between empty vector and 6E-PEST groups.</p

Physico-chemical properties of scFv-C4 intrabodies.

<p>Physico-chemical determinants of soluble intrabody expression. Solubility is improved by an overall negative charge at pH 7.4 and reduced GRAVY score. The GRAVY score is the average hydropathy score for all the amino acids in the protein, with a more negative score indicating increased hydrophilicity. The PEST-FIND program <a href="http://emboss.bioinformatics.nl/cgi-bin/emboss/pestfind" target="_blank">http://emboss.bioinformatics.nl/cgi-bin/emboss/pestfind</a> objectively produces a score ranging from about −50 to +50. A score above +5 denotes a PEST region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#pone.0029199-Rechsteiner1" target="_blank">[33]</a>.</p

Scrambling the PEST sequence and specific inhibition of the proteasome eliminates the enhanced degradation of httex1-72Q-GFP.

<p><b>A</b>. 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>B</b>. Biochemical fractionation of HA-tagged fluorobodies from ST14A cells. Detergent-soluble and -insoluble cell lysates were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#s4" target="_blank">Materials and Methods</a>. 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 <b>C</b>. AGERA. There is an increase of detergent-insoluble material in scFv-C4-PEST-SCR transfected cells compared to scFv-C4-PEST. <b>D</b>. 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 <b>E</b>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#pone.0029199-Meng1" target="_blank">[34]</a>; 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.</p

PEST fusion reduces steady-state scFv-C4 intrabody levels modestly, and independent of antigen.

<p>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.)</p

Bifunctional nanobody-PEST reductions of α-Syn levels.

<p>(A) Dual transfection of wild type α-Syn plus VH14 constructs in ST14A cells. VH14PEST (VH14P) and VH14 with a scrambled PEST degron (VH14S) significantly reduced (*p<0.05, n = 5) α-Syn based on Western blot densitometry of α-Syn compared with VH14 and empty vector control. (B) Endogenous α-Syn after transfection of VH14PEST constructs in SH-SY5Y cells. Western blot densitometry shows that α-Syn is significantly (*p<0.05, n = 4) reduced by VH14P compared with empty vector control, VH14 and VH14S control (C) Live cell imaging, Western blot, and densitometry for Syn with anti-Syn1. Syn87PEST (Syn87P) significantly reduced empty vector control Syn~GFP protein levels compared to VH14P and Syn2P (Mean ± SEM. *p<0.05 compared to Control and Syn87PEST; ᵻ p<0.05 compared to all groups; n = 3).</p

VH14PEST decreases the toxicity of α-Syn as measured by a proteostatic assay with mhttex1-72QGFP.

<p>VH14PEST reduced toxicity induced by proteostatic stress of co-expressing mhttex1-72Q-GFP with WT α-Syn overexpression. Overexpression of empty vector control plus mhttex1-72Q-GFP and WT Syn significantly increased early apoptotic cell death compared to empty vector GFP control [Mean ± SEM. *p<0.05 significantly different from all groups; n = 3]. VH14PEST significantly (‡p<0.05) reduced the percentage of apoptotic cell death to a greater extent than Syn87PEST [Mean ± SEM. *p<0.05 compared to Empty Vector control GFP; ‡p<0.05 comparing VH14PEST vs. Syn87PEST; †p<0.05 comparing Syn87PEST vs empty vector control-Syn-mhttex1-72Q-GFP.]. Lower Panel: Representative scatter plots showing the percentage of cells in each quadrant.</p

Overexpression of Syn87PEST and VH14PEST reduced total cell death to control levels in ST14A cells.

<p>(A) VH14PEST reduced the percentage of total cell death to a greater extent than Syn87PEST. [Mean ± SEM. * p<0.05 compared to Syn-GFP; † p<0.05 compared to Syn87PEST; n = 3]. (B & C) Representative scatter plots showing the percentage of cells in each quadrant. Viable cells are annexin V (−) and 7-AAD (−); annexin V (+) and 7-AAD (−) cells are in early apoptosis; annexin V (+) and 7-AAD (+) cells are in late apoptosis; necrotic cells are annexin V (−) and 7-AAD (+). (B) The percent total of cells undergoing late apoptosis was significantly reduced by VH14PEST compared to empty vector control co-transfected GFP levels. (Mean ± SEM. *p<0.05 comparing control GFP, to other groups; ᵻ p<0.05 compared VH14PEST/SYNGFP to other groups; n = 3). (C) The percent Annexin V positive cells undergoing early apoptosis was significantly reduced to empty vector control co-transfected GFP levels. (Mean ± SEM. *p<0.05 comparing control GFP, VH14PEST Syn-GFP to other groups; n = 3).</p

GFP expression in lumbar spinal cords of NHPs monitored by IHC.

<p>GFP expression in lumbar spinal cords of NHPs monitored by IHC.</p