Tauseef R. Butt

Regulation of human estrogen receptor by phytoestrogens in yeast and human cells

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estrogen. They are present in many foods and their higher consumption in certain populations has been correlated with protection against many diseases including coronary heart disease, breast cancer and endometrial and ovarian cancer. In this report, ten phytoestrogens with diverse chemical structures were studied for their binding to the human estrogen receptor and their transcription activation properties in yeast and mammalian cells. Our results showed that some of these compounds bind with relatively high affinity to the estrogen receptor and activate the receptor in the yeast and mammalian cell system. In addition, none of these compounds showed anti-estrogenic activity. We conclude that the yeast system accurately predicts the estrogenic activity of compounds with diverse chemical structures in mammalian cells. In addition, our data with phytoestrogens that do not show transcription activation properties raise the possibility that these compounds may exert their biological effects through pathways different from the classical estrogen signalling mechanism.

Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists.

An expression system that utilized yeast copper metallothionein promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter beta-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.

Structural and functional analysis of N-terminal point mutants of the human estrogen receptor

Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.

Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin

SummaryLibraries of random-sequence polypeptides have been shown to be valuable sources of novel molecules possessing a variety of useful biologic-like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced inE. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.

Protein Folding Absent Selection

Biological proteins are known to fold into specific 3D conformations. However, the fundamental question has remained: Do they fold because they are biological, and evolution has selected sequences which fold? Or is folding a common trait, widespread throughout sequence space? To address this question arbitrary, unevolved, random-sequence proteins were examined for structural features found in folded, biological proteins. Libraries of long (71 residue), random-sequence polypeptides, with ensemble amino acid composition near the mean for natural globular proteins, were expressed as cleavable fusions with ubiquitin. The structural properties of both the purified pools and individual isolates were then probed using circular dichroism, fluorescence emission, and fluorescence quenching techniques. Despite this necessarily sparse “sampling” of sequence space, structural properties that define globular biological proteins, namely collapsed conformations, secondary structure, and cooperative unfolding, were found to be prevalent among unevolved sequences. Thus, for polypeptides the size of small proteins, natural selection is not necessary to account for the compact and cooperative folded states observed in nature.

A Disposable Cell-Based Biochip for Detection of Hormones and Drugs

This paper presents development and characterization of a disposable cell-based biochip for detection of hormone dependent responses of human nuclear receptors. The disposable cell-based biochip has chamber arrays for immobilize cell-based sensors and microfluidic channels for hormone and substrate injection. A prototype plastic microarray has been designed, fabricated, and validated for hormone dependent, cell-based, human nuclear receptor function. Depending upon the receptor employed, these sensors can determine predisposition to type II diabetes and cardiovascular diseases.