Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity

Successful design of tissue engineering scaffolds must include the ability to stimulate vascular development by incorporating angiogenic growth factors. Current approaches can allow diffusion of growth factors, incorporate active factors randomly, or can leave residual toxins. We addressed these problems by genetically fusing the gene encoding Vascular Endothelial Growth Factor (VEGF) with the Ultrabithorax (Ubx) gene to produce fusion proteins capable of self-assembly into materials. We demonstrate that VEGF-Ubx materials enhance human endothelial cell migration, prolong cell survival, and dose-dependently activate the VEGF signaling pathway. VEGF-Ubx fibers attract outgrowing sprouts in an aortic ring assay and induce vessel formation in a chicken embryo chorioallantoic membrane (CAM) assay. Collectively, these results demonstrate that the activity of VEGF remains intact in Ubx materials. This approach could provide an inexpensive and facile mechanism to stimulate and pattern angiogenesis

The intrinsically disordered regions in Ubx are necessary for protein interactions.

<p>Yeast two-hybrid indicates that Ubx variants, either lacking all disordered regions (UbxIVa N216) or all structured regions (UbxIb N103 Δ292–389 Pro4 mCherry), cannot bind partner proteins. Likewise, AkUbx, a primitive Ubx orthologue derived from <i>Acanthokara kaputensis</i>, naturally lacks most of the disordered sequences and is also unable to bind partner proteins.</p

Ubx variants did not interact with B42 protein activation in the absence of Ubx partners.

<p>Yeast two-hybrid results for wild type full length Ubx or Ubx variants with truncation and/or Pro4 mutation showed no significant interaction with B42 protein activation domain from β-galactosidase reporter gene expression, listed as Miller Units.</p

Specific folds are enriched in Ubx-binding proteins.

<p>Exd is a well-established Ubx binding protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Johnson1" target="_blank">[50]</a>, and Ubx cooperatively binds DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Beachy1" target="_blank">[77]</a>. All other Ubx binding partners were identified by yeast two-hybrid assays. Ubx binding partners were classified by the fold/shape according to SCOP. Folds with more than one partner were defined as “selected”. The interactions with Term, Fzo, mRpL44, and Pk17E were reported by Giot <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Giot1" target="_blank">[51]</a>. The remaining interactions were reported by Bondos <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Bondos2" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Bondos3" target="_blank">[49]</a>. Ubx binding proteins with non-selected folds are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217.s006" target="_blank">Table S1</a>.</p><p>Specific folds are enriched in Ubx-binding proteins.</p

Ubx splicing isoforms are differentially able to bind partner proteins.

<p>Whereas all partners with an α-α superhelix fold bind UbxIa better than UbxIVa, among partners with a DNA/RNA binding 3-helical bundle fold only Al binds these two Ubx isoforms differently. “Inc” denotes Region 3, the microexon region, and is incomplete in the UbxIa splicing isoform. The disordered regions remaining in each variant are listed in parentheses after the protein name. (B) Colored lines represent intrinsic disorder prediction scores for the microexon region for different Ubx splicing isoforms, generated using the PONDR VL-XT algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Li1" target="_blank">[89]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Romero1" target="_blank">[90]</a>. Blue line, UbxIb which has all 3 microexons; grey line, UbxIa which lacks the 9 amino acid b element; red line, UbxIVa, which lacks all 3 microexons. Dashed lines connect data across the microexon sequences removed in the shorter isoforms. The extent of predicted disorder (score>0.6, region shaded light grey) correlates with the ability to bind the tested partner proteins.</p

A comparison of the occurrence of folds in the <i>Drosophila</i> proteome and interactome.

<p>p-value of enriched fold relative to <i>Drosophila</i> proteome/interactome was generated using Chi-Squared test.</p><p>A comparison of the occurrence of folds in the <i>Drosophila</i> proteome and interactome.</p

Ubx recognizes three categories of DNA binding sites.

<p>Ubx cooperatively binds multimers of Hox binding sites (TAAT/ATTA, red text), including enhancers for the <i>ubx</i> and <i>antp</i> genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Jemth1" target="_blank">[76]</a>. Other transcription factors are not known to influence Ubx binding to these sites. In the second category, DNA binding sites for Ubx monomers are separated by DNA binding sites for other transcription factors (Medea, purple text, and Mad, green text) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Walsh1" target="_blank">[91]</a>. Regulation of the <i>sal</i> gene is coordinated by both Ubx and BMP signaling, which controls the activity of Medea and Mad. In the final category, Ubx binds DNA and regulates transcription in association with Exd (blue text) and Hth (orange text), general Hox co-factors <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Vachon1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Gebelein1" target="_blank">[93]</a>. The positions of the DNA sequences are marked in bp relative to the start of transcription.</p

Location of structured and disordered regions in UbxIb, and design of Ubx variants.

<p>(A) A grey bar, representing the domain organization of the UbxIb transcription factor shows the position of its transcription activation domain (blue), YPWM Exd interaction motif (yellow), DNA-binding homeodomain (black), a partial transcription repression domain (orange), and protein regions encoded by three alternatively spliced microexons: the b element (pink), mI (purple), and mII (brown). (B) The location of predicted protein-interaction motifs in Ubx as predicted by ANCHOR (yellow stripes) and MoRFpred (blue stippled stripes). Regions predicted by both algorithms to be involved in protein interactions are marked with both yellow and blue. (C) A bar schematic depicting the positions of structured and intrinsically disordered regions in UbxIb. The boundaries were determined by a combination of computational and experimental approaches. The scores from three disorder prediction algorithms were averaged to identify structured (green) and disordered (red) regions. Native state proteolysis, in which only disordered segments can be cleaved by trypsin, was used to verify these assignments, and, where appropriate, slightly expanded the boundaries of the predicted disordered regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Liu1" target="_blank">[6]</a>. Sites cut by trypsin (black triangles), sites not cut by trypsin (open triangles), and sites that could not be definitively assigned (grey triangles) are indicated. (D) Bar schematic for predicted protein interfaces and molecular recognition features (MoRFs) on Ubx peptide. The schematic bars show Anchor algorithm predicted Ubx- partner protein interfaces (orange bars) and MoRF algorithm predicted Ubx-partner protein interface (blue bars with pattern fill). (D) Bar schematics of Ubx truncation mutants and internal deletion mutants used in yeast two-hybrid assays to identify partner binding interfaces. UbxIb, UbxIa, and UbxIVa are isoforms created by alternative splicing <i>in vivo</i>. To prevent auto-activation, the activation domain was de-activated either by removal of amino acids 102 to 216 or by the Pro4 mutation, in which Ala and Glu are mutated to Pro at amino acids 226 and 233 (indicated by a red-green stipple), respectively, which should prevent formation of a predicted α-helix required for transcription activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108217#pone.0108217-Tan1" target="_blank">[43]</a>. In two variants, the structured C-terminus of the protein was replaced by mCherry, represented by a pink/white striped bar.</p

An individual partner domain is sufficient for Ubx binding.

<p>Full length Al and Arm have similar interaction strength as individual domains derived from Al (residues 81–142) and Arm (residues 155–273) with UbxIb Pro4. The intensity of the β-galactosidase reporter gene, reported as Miller Units, signal for each partner is similar to its respective single-domain variant.</p

Partner proteins bind more than one region in Ubx.

<p>Yeast two-hybrid results for Ubx variants in which each region of Ubx has been sequentially mutated or deleted. Each of these variants retain some ability to bind Ubx relative to UbxIbN216 (0,0, S3, D4, S5). Partners are grouped based on the fold they have in common.</p