Fig 1.
Simvastatin pro-drug compared with its hydrolysis product and pravastatin.
Fig 2.
Immobilisation of a small molecule active using multiple photochemistries.
The use of five different photochemistries in a multiwall format allows the simultaneous, rapid exploration of several regio- and other isomeric derivatives.
Fig 3.
Preparation of a T7 phage display library from vascular tissue.
Fig 4.
Ethidium bromide stained agarose gel showing a sample of clones from the phage display library after PCR to amplify the insert DNA.
Hyperladder in lanes 1 and 25 allows estimation of insert length. PCR controls in lanes 22, 23 and 24 (positive, positive and negative).
Fig 5.
Biopanning against the genomic phage library displaying 5–15 polypeptide copies per headgroup.
Elution is carried out herein using the host bacterium, E. coli BLT5615 avoiding potential inhibitory or other effects of an active small molecule.
Fig 6.
Alignment of discovered contig29 versus GJC3 (residues 1–25) and the sequence for GJB2 (residues 1–25).
Carried out using T-Coffee (www.ebi.ac.uk) with GJC3 NCBI sequence NP_853516.1 and the protein databank file corresponding to GJB2 code 2zw3.pdb. Consensus residues are shown in the upper row; figure prepared using UCSF Chimera [50].
Fig 7.
Comparison of Kyte-Doolittle index hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive values.
Kyte-Doolittle analysis using ProtScale [124] http://web.expasy.org/protscale/. Parameters used were: window size = 9, relative weight 100%, linear model, not normalized. Data replotted using ggplot2 in R [125].
Fig 8.
Wimley-White hydropathy plot of GJC3 (orange) and GJB2 (blue) showing predicted transmembrane regions as positive negative values.
Wimley-White octanol-to-water scale smoothed values for GJC3 and GJB2 are shown, where positive hydropathy values indicate expected membrane associated residues. The predicted transmembrane (TM) segments indicated are at least 19 residues in length and were calculated using a validated algorithm [126]. Wimley-White smoothed hydropathy calculated using MPEx [127] http://blanco.biomol.uci.edu/mpex/ and replotted using ggplot2 in R [125].
Fig 9.
Discovered N-terminal residues 1–25 (orange) aligned on a simple model based on GJB2.pdb X-ray structure.
Hexameric structure viewed from cytoplasmic side of the transmembrane hemichannel (prepared using UCSF Chimera [50]).
Fig 10.
Immunohistochemistry for human artery with antibody against GJC3, hCx30.2/31.3.
(A) H-86—sc-68376, Santa Cruz Biotechnology, Inc., vs. (B) control section, both micrographs at 20 x magnification. Brown staining in (A) indicates selective localisation of GJC3 in media and endothelium of the artery.
Fig 11.
Alternative mRNA splicing and selected single nucleotide polymorphisms for GJC3 products.
CCDS entry CCDS34697.1 (accessed 24 May 2012) for transcribed mRNA from GJC3 (NM_181538.2 and NP_853516.1) and its translated 279 amino acid polypeptide showing the alternate exon region, designated with a blue rectangle. Residues highlighted in purple (M1V, R4V, P19H, R22H, E268D) indicate single nucleotide polymorphisms (SNPs: see main text) leading to variation in coded amino acids within predicted geometrically neighbouring and functionally significant N- and C-terminal regions. V261 highlighted in orange indicates the C-terminal splice variant start point. Discovered residues correspond to amino acids 1–25. Underlined residues S227S, T234N highlighted in green indicate phosphorylation sites identified from PhosphositePlus and genomic SNPs.
Fig 12.
Surface plasmon resonance sensorgrams of peptide A interacting with statins.
(A) Simvastatin hydroxyacid, (B) simvastatin lactone, (C) fluvastatin hydroxyacid, and (D) pravastatin hydroxycid, each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent interactions are observed for both simvastatin and fluvastatin hydroxyacid sodium salts with the most pronounced interaction observed for fluvastatin.
Fig 13.
Surface plasmon resonance sensorgrams of peptide A interacting with known hemichannel blockers.
(A) Niflumic acid, (B) flufenamic acid, (C) carbenoxolone, and (D) 18β-glycyrrhetinic acid each at concentrations of 400 μM (light blue), 200 μM (red), 100 μM (grey), 50 μM (yellow), 25 μM (dark blue) 0 μM (green) for each ligand in PBS. Concentration dependent binding profiles are observed for niflumic acid with more pronounced effects observed for flufenamic acid and carbenoxolone.
Fig 14.
Chemical structures of enoxolone (18β-glycyrrhetinic acid), 18α-glycyrrhetinic acid) and carbenoxolone (18β-glycyrrhetinic acid).
Fig 15.
Helical wheel projections of: (A) discovered N-terminal residues 1–25 and (B) C-terminal residues 226–227.
Serine 227 and threonine 234 subject to post-translational phosphorylation circled in red.