Figure 4. Identification of the amyloid converting motif that targets proteins to A-bodies.

(A) VHL can obtain an amyloid-like conformation in bacteria. GFP or VHL-GFP expressing MCF-7 cells left untreated or exposed to acidosis or BL21 cells were stained with Congo red and Hoechst. X-ray diffraction was performed on BL21 bodies. (B) VHL contains fibril-forming peptidic regions. Results of ZipperDB analysis of full length VHL for fibrillation propensity. The Rosetta energy threshold of −23 kcal/mol is an indicator of fibril-positive regions. Truncated VHL fragments, used below, are indicated. (C) Amyloidogenic fragments of VHL insolubilize GFP and produce SDS-resistant multi-mers. Fragments of VHL (above) fused to GFP were expressed in BL21, prior to lysis and insoluble protein fractionation. Fusion proteins were detected with a GFP-specific antibody at low and high exposures to detect monomeric (low) and multi-meric (high) proteins. (D) Insoluble VHL fragments form bacterial inclusion bodies with an amyloid-like x-ray diffraction profile. BL21 expressing the VHL fragments (above) were fixed and stained with Congo red and Hoechst. Inclusion bodies were purified, where present, for x-ray diffraction. (E) Table summarizing inclusion body formation and targeting to the A-bodies (Figure 4D, S4B, F and H) for the indicated regions of VHL, cdk1 or POLD1 fused to GFP. (F) Regions of VHL can associate with rIGS₂₈RNA. MCF-7 cells transfected with VHL or VHL mutants were exposed to acidosis for 1 hour prior to lysis and RNA immunoprecipitation. 28S rRNA and rIGS₂₈RNA were detected by RT-PCR. Exogenous VHL fragments and GAPDH were detected by immunoblotting. (G) Generation of artificial ACM sequences. R/H-rich (orange) and amyloidogenic (purple) sequences derived from VHL (aACM^VHL) and POLD1 (aACM^POLD1) were fused to generate artificial ACM motifs (sequence inset). The fibrillation propensity was calculated by ZipperDB. (H) Artificial ACMs are sufficient to create insoluble multi-mers. BL21 expressing GFP fused to the artificial ACMs (above) were lysed into soluble (+) and insoluble (−) fractions. (I) Amyloidogenic regions of VHL form 10nm amyloid-like fibrils. Peptides encoding VHL (104–140), aACM^VHL and the classic pathological β-amyloid were synthesized and incubated for 1 week at 37°C. Fibrils were detected by TEM. White and yellow scale bars represent 20µm and 5µm, respectively. Dashed circles represent nuclei (MCF-7) or whole cell (BL21).TEM scale box represent 10nm. See also Figure S4.