Rationally modified estrogen receptor protein as a bio-recognition element for the detection of EDC pollutants: strategies and opportunities

The estrogen receptor protein (ER) can bind a vast number of organic pollutants widely spread in the environment and collectively known as Endocrine Disrupting Chemicals, EDCs. Its broad selectivity makes it an ideal bio-recognition element for the detection of EDCs. Here we describe the strategy and rationale for the design of ER based biosensors and assays that generate a signal in the presence of EDCs. The opportunity to use either natural or rationally modified ER molecules is discussed. The latter approach was successfully applied in the EU-FP7 project RADAR, with the aim to develop a novel biosensor for the detection of organic pollutants both in the environment and in commercial water products

Rational modification of estrogen receptor by combination of computational and experimental analysis

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor- ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC50) value of 2 nM for the 17α- ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor

Rational modification of estrogen receptor by combination of computational and experimental analysis.

In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC50) value of 2 nM for the 17α-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor

Schematic representation of the structural domains of ER protein.

<p>A) A cartoon representation of the three-dimensional structure of the ligand binding domain is shown, as well as its sequence. Residues belonging to the ligand binding pocket are shown in red in both the structure and sequence. Residues highlighted in yellow belong to the histidine tag, residues in light blue encompass the ligand binding domain. B) Degree of conservation for residues of the ligand binding pocket among the analyzed ER sequences. Full bars correspond to 100% conservation.</p

IC₅₀ values and standard errors resulting from the competitive binding assay performed in four replicates with wt-ER_α^LBD (column 2), M421F- ER_α^LBD (column 3) and M421I- ER_α^LBD (column 4) and selected compounds (ligands, column 1): 17β-estradiol, 17α-ethinylestradiol, bisphenol-A, tamoxifen, 4-nonylphenol and 4-tert-octylphenol.

<p>The M421F-ER_α^LBD shows lower IC₅₀, compared to wt-ER_α^LBD, for four compounds (17β-estradiol, 17α-ethinylestradiol, bisphenol-A and 4-nonylphenol) out of six.</p><p>The M421I-ER_α^LBD has higher IC₅₀, compared to wt-ER_α^LBD, for all the tested compounds.</p

Circular dichroism and SDS-PAGE of recombinant ER proteins.

<p>CD spectra show typical α-helical character and a single band at the expected molecular weight is visible (insets). (A) Far-UV CD and SDS-PAGE of wt-ER_α^LBD (B) Far-UV CD and SDS-PAGE of M421F-ER_α^LBD (C) Far-UV CD and SDS-PAGE of M421I-ER_α^LBD (D) – (F): thermal unfolding data of the three proteins. Intensity of peak at 222 nm plotted as a function of increased temperature. Melting temperature (T_m) reported.</p

Competitive binding assay.

<p>Competitive binding assay on wt-ER_α^LBD (blue squares), M421F-ER_α^LBD (red circles) and M421I-ER_α^LBD (green triangles) with six different compounds: 17β-estradiol (panel A), 17α-ethinylestradiol (panel B), bisphenol-A (panel C), tamoxifen (panel D), 4-nonylphenol (panel E) and 4-tert-octylphenol (panel F).</p

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue1, 2. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat–containing protein that positively modulates RAS-MAPK signal flow3, 4, 5, 6, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721)7 shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2Gin vitro enhanced MAPK activation in a cell type–specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease