O'Hara, Patricia B. 2012 Biphysical

Estrogen receptors (ERα and ERβ) are ligand-binding transcription factors activated by the hormone 17-β estradiol. Ligand binding triggers ER dimerization, translocation of the receptor from the cytosol into the nucleus and eventually activation of the genes under control of ER. Studies have revealed a role for estrogen receptors in male and female sexual development, reproductive functions, bone metabolism and regulation of neuroendocrine and cardiovascular systems. ER is also known to bind to other nonnative ligands known in pharmacology as receptor agonists or antagonists. Agonists provoke a biological response when bound to the receptor; antagonists inhibit a biological response when bound. Our lab is interested in the promiscuous binding of the estrogen receptor and its ability to activate different genes in different tissues. Fluorescence polarization assays have previously been performed using ER to study ligand binding affi nities for the receptor. However, this technique is unable to determine whether these ligands are agonists or antagonists and allow ER dimerization and gene activation. To investigate these phenomena, an activity assay that measures ER controlled gene expression has been developed which provides the opportunity to gain further insight into the functional activity in living systems. Recombinant yeast cells that express ERα use the green fl uorescent protein (GFP) reporter to determine whether ERα, in the presence of a particular ligand, has activated gene of expression. We have correlated the binding to agonist and antagonist behavior of several xeno- and phyto- estrogens.

α-Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α-Crystallin (αX) oligomers consist of two isoforms, αA and αB which share high sequence similarity and define the common α-Crystallin fold found in many small heat shock proteins (sHSPs). αA and αB are hypothesized to play two important roles within the lens. First, αA and αB belong to a group of proteins called Crystallins (α, β, and γ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this highdensity protein organ. Second, αA and αB both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between αA and αB that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of αA and αB to simulate long-term protein damage and degradation observed in aged lenses. We hope to identify specific molecular interactions that result in αA and αB’s chaperone function, and determine how those interactions relate to stability and selfoligomerization.