Data from: Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it

Total proteome turbidity assay for tracking global protein aggregation in the natural cellular environment

Proteome homeostasis is crucial for optimal cellular function and survival in the face of various stressful impacts. This entails preservation of a balance between protein synthesis, folding, degradation, and trafficking collectively termed proteostasis. A hallmark of proteostasis failure, which underlies various diseases, is enhanced misfolding and aggregation of proteins. Here we adapted the measurement of protein turbidity, which is commonly used to evaluate aggregation of single purified proteins, for monitoring propensity for aggregation of the entire soluble cellular proteome incubated in vitro for several hours. We show that over-expression of an aggregation-prone protein or applying endoplasmic-reticulum (ER) stress to either cells in culture or to the intact organism, Drosophila, enhances the rise in turbidity of the global soluble proteome compared to untreated cells. Additionally, given that Alzheimer’s disease (AD) is known to involve ER stress and aggregation of proteins, we demonstrate that the soluble fraction of brain extracts from AD patients displays markedly higher rise of global proteome turbidity than in healthy counterparts. This assay could be valuable for various biological, medical and biotechnological applications

Data from: Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it

dVHLModel

Coordinate file (PDB format) of the model structure of the Drosophila VHL protein

Analysis from k2d2 program of pVHL and dVHL suggest similar secondary structure content of α-helix, β-sheet and random structure.

<p>Analysis from k2d2 program of pVHL and dVHL suggest similar secondary structure content of α-helix, β-sheet and random structure.</p

The GFP fluorescence signal caused by over-expression of ODD-GFP is lowered by co-expression of either dVHL, pVHL19, or pVHL30.

<p>(<b>A</b>) <b>GFP levels</b> were analyzed by confocal microscopy. Genotypes: GMR-Gal4; UAS-ODD-GFP/UAS-RFP (left), GMR-Gal4; UAS-ODD-GFP/UAS-dVHL (second from left) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL19 (third from the left) and GMR-Gal4; UAS-ODD-GFP; UAS-pVHL30 (right). Scale bar, 20 µm (<b>B) Western blot</b> analysis showing the levels of ODD-GFP protein extract from flies heads. β-actin was used as loading control. (<b>1</b>) GMR-Gal4; UAS-ODD-GFP/UAS-RFP. (<b>2</b>) GMR-Gal4. (<b>3</b>) GMR-Gal4; UAS-ODD-GFP/UAS-dVHL. (<b>4</b>) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL19. (<b>5</b>) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL30.</p

Subcellular localization of pVHL in transgenic Drosophila.

<p>Immunofluorescence staining of the <i>Drosophila</i> eye imaginal discs over expressing pVHL30 by GMR Gal4. (<b>A</b>) Bright field (<b>B</b>) Nuclei stained using DAPI (blue) (<b>C</b>) Actin stained using phalloidin 568 (red) (<b>D</b>) Anti-pVHL antibody was used for identification of pVHL protein (purple). (<b>E</b>) Merge. Upper panel scale bar: 20 µm; lower panel scale bar: 10 µm (zoom in).</p

Sequence alignment of pVHL and dVHL, the alignment was generated using the MUSCLE algorithm, and shows structural motifs (β strands and α helices) of pVHL and conservation of the Cul2 and BC boxes as well as residues involved in Hif-1α binding.

<p>*(asterisk) - fully conserved residue: (colon) - similar residue, scoring >0.5;. (period) weakly similar residue, scoring = <0.5.</p

Folding, stability and function of VHL monitoring by biophysical methods.

<p>(<b>A, B</b>) <b>Circular dichroism:</b> Far-UV (A) and near-UV (B) CD spectra of pVHL (blue) and dVHL (red). CD spectra measurements were conducted at 25°C. Protein sample was at final concentration of 5 µM in 10 mM Tris-HCl (pH 8) and 200 mM NaCl. (<b>C, D</b>) <b>Size exclusion chromatography:</b> preformed at 10 mM Tris-HCl (Ph 8) and 200 mM NaCl. (C) Calibration curves generated for molecular weight standards (Kav versus Log MW). Apparent molecular mass of pVHL (blue) and dVHL (red) was calculated with the curve equation (C). Apparent R_S of pVHL (blue) and dVHL (red) were calculated with the curve equation (√-logKav versus R_S) (D). Molecular weight standard proteins (black): Vitamin B12 (1.35 kDa, 0.75 nm), Myoglobin (17 kDa, 1.9 nm), Ovalbomin (44 kDa, 2.8 nm), γ-globulin (158 kDa, 5.1 nm) and Thyroglobulin (670 kDa, 8.6 nm). (<b>E</b>) <b>ANS fluorescence studies:</b> Comparison of spectrum measured with ANS alone (blue), and spectrum obtained following addition of 3 µM dVHL at different temperatures (4, 25, 37 and 50°C) and after cool down (E). (<b>F</b>) <b>Functional analysis of dVHL and pVHL:</b> Binding of dVHL or pVHL to TAMRA labeled Hyp402-ODD-HIF or Hyp850-ODD-SIMA target peptides (F). Fluorescence of TAMRA at 580 nm was measured as a function of log of peptide concentration (mM). The results are presented after subtraction of the negative control (BSA with peptide at different concentrations). pVHL or dVHL protein concentration was 500 nM; pVHL incubated with Hyp402-ODD-HIF peptide (blue), pVHL incubated with Hyp850-ODD-SIMA peptide (red), dVHL incubated with Hyp402-ODD-HIF peptide (green) and dVHL incubated with Hyp850-ODD-SIMA peptide (purple).</p