Bahareh Azizi

Fluorescence imaging using synthetic GFP chromophores

Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.

Histone Deacetylase Inhibitors Equipped with Estrogen Receptor Modulation Activity

We describe a set of novel histone deacetylase inhibitors (HDACi) equipped with either an antagonist or an agonist of the estrogen receptor (ER) to confer selective activity against breast cancers. These bifunctional compounds potently inhibit HDAC at nanomolar concentrations and either agonize or antagonize ERα and ERβ. The ER antagonist activities of tamoxifen-HDACi conjugates (Tam-HDACi) are nearly identical to those of tamoxifen. Conversely, ethynyl-estradiol-HDACi conjugates (EED-HDACi) have attenuated ER agonist activities relative to the parent ethynyl-estradiol. In silico docking analysis provides structural basis for the trends of ER agonism/antagonism and ER subtype selectivity. Excitingly, lead Tam-HDACi conjugates show anticancer activity that is selectively more potent against MCF-7 (ERα positive breast cancer) compared to MDA-MB-231 (triple negative breast cancer), DU145 (prostate cancer), or Vero (noncancerous cell line). This dual-targeting approach illustrates the utility of designing small molecules with an emphasis on cell-type selectivity, not merely improved potency, working toward a higher therapeutic index at the earliest stages of drug development.

Characterization of a molecular switch system that regulates gene expression in mammalian cells through a small molecule

BackgroundMolecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Nuclear receptors are valuable candidates for these regulation systems due to their functional role as ligand activated transcription factors. Previously, our group engineered a variant of the retinoid × receptor to be responsive to the synthetic compound, LG335, but not responsive to its natural ligand, 9-cis-retinoic acid.ResultsThis work focuses on characterizing a molecular switch system that quantitatively controls transgene expression. This system is composed of an orthogonal ligand/nuclear receptor pair, LG335 and GRQCIMFI, along with an artificial promoter controlling expression of a target transgene. GRQCIMFI is composed of the fusion of the DNA binding domain of the yeast transcription factor, Gal4, and a retinoid × receptor variant. The variant consists of the following mutations: Q275C, I310M, and F313I in the ligand binding domain. When introduced into mammalian cell culture, the switch shows luciferase activity at concentrations as low as 100 nM of LG335 with a 6.3 ± 1.7-fold induction ratio. The developed one-component system activates transgene expression when introduced transiently or virally.ConclusionsWe have successfully shown that this system can induce tightly controlled transgene expression and can be used for transient transfections or retroviral transductions in mammalian cell culture. Further characterization is needed for gene therapy applications.

A human vitamin D receptor mutant activated by cholecalciferol.

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.

Conservation of estrogen receptor function in invertebrate reproduction

BackgroundRotifers are microscopic aquatic invertebrates that reproduce both sexually and asexually. Though rotifers are phylogenetically distant from humans, and have specialized reproductive physiology, this work identifies a surprising conservation in the control of reproduction between humans and rotifers through the estrogen receptor. Until recently, steroid signaling has been observed in only a few invertebrate taxa and its role in regulating invertebrate reproduction has not been clearly demonstrated. Insights into the evolution of sex signaling pathways can be gained by clarifying how receptors function in invertebrate reproduction.ResultsIn this paper, we show that a ligand-activated estrogen-like receptor in rotifers binds human estradiol and regulates reproductive output in females. In other invertebrates characterized thus far, ER ligand binding domains have occluded ligand-binding sites and the ERs are not ligand activated. We have used a suite of computational, biochemical and biological techniques to determine that the rotifer ER binding site is not occluded and can bind human estradiol.ConclusionsOur results demonstrate that this mammalian hormone receptor plays a key role in reproduction of the ancient microinvertebrate Brachinous manjavacas. The presence and activity of the ER within the phylum Rotifera indicates that the ER structure and function is highly conserved throughout animal evolution.

The role of residue C410 on activation of the human vitamin D receptor by various ligands.

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in biologically important processes. The human vitamin D receptor (hVDR) is a member of the NR superfamily and is responsible for maintaining calcium and phosphate homeostasis. This receptor is activated by its natural ligand, 1α, 25-dihydroxyvitamin D(3) (1α, 25(OH)(2)D(3)), as well as bile acids such as lithocholic acid (LCA). Disruption of molecular interactions between the hVDR and its natural ligand result in adverse diseases, such as rickets, making this receptor a good target for drug discovery. Previous mutational analyses of the hVDR have mainly focused on residues lining the receptors ligand binding pocket (LBP) and techniques such as alanine scanning mutagenesis and site-directed mutagenesis. In this work, a rationally designed hVDR library using randomized codons at selected positions provides insight into the role of residue C410, particularly on activation of the receptor by various ligands. A variant, C410Y, was engineered to bind LCA with increased sensitivity (EC(50) value of 3 μM and a 34-fold activation) in mammalian cell culture assays. Furthermore, this variant displayed activation with a novel small molecule, cholecalciferol (chole) which does not activate the wild-type receptor, with an EC(50) value of 4 μM and a 25-fold activation. The presence of a bulky residue at this position, such as a tyrosine or phenylalanine, may contribute towards molecular interactions that allow for the enhanced activation with LCA and novel activation with chole. Additional bulk at the same end of the pocket, such as in the case of the variant H305F; C410Y enhances the receptors sensitivity for these ligands further, perhaps due to the filling of a cavity. The effects of residue C410 on specificity and activation with the different ligands studied were unforeseen, as this residue does not line the hVDRs LBP. Further investigating of the structure-function relationships between the hVDR and its ligands, including the mutational tolerance of residues within as well as outside the LBP, is needed for a comprehensive understanding of the functionality and interactions of the receptor with these ligands and for development of new small molecules as potential therapeutic drugs.

BAPJ69-4A: A yeast two-hybrid strain for both positive and negative genetic selection

Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein-protein and protein-ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering.