Peptide-conjugated glass slides for selective capture and purification of diagnostic cells: Applications in urine cytology.

Obtaining a clear view of the cells of interest in diagnostic cytology can be challenging when specimens are contaminated with blood or other obscuring cells. In this study, we present a powerful technique for the selective capture of diagnostic epithelial cells directly on a microscope slide, highlighting its applications in urine cytology and immunocytochemistry (ICC). Using phage-display biopanning, we identified and synthesized a series of peptides that bind with high affinity to urothelial cells but not blood cells. We developed methods for conjugating the peptides to glass slides, and we used these slides to selectively capture both normal and cancerous epithelial cells from urine contaminated with blood cells. Unlike non-selective microscope slides, the peptide-conjugated slides selectively retained the cells of interest, recovering up to 75% of urothelial cells, while up to 98% of blood cells were washed away. The slides are compatible with Papanicolaou and hematoxylin and eosin (H&E) staining for cytology preparations, as well as ICC for detecting membrane-associated and nuclear cancer markers. We successfully detected the expression of carcinoembryonic antigen and survivin, two commonly measured bladder cancer markers. In addition to bladder cancer diagnostics, this technology has broad applications for increasing the quality of sample preparations in slide-based diagnostic testing.

Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands.

The pharmacological activity of different nuclear receptor ligands is reflected by their impact on receptor structure. Thus, we asked whether differential presentation of protein-protein interaction surfaces on the androgen receptor (AR), a surrogate assay of receptor conformation, could be used in a prospective manner to define the pharmacological activity of bound ligands. To this end, we identified over 150 proteins/polypeptides whose ability to interact with AR is influenced in a differential manner by ligand binding. The most discriminatory of these protein-AR interactions were used to develop a robust compound-profiling tool that enabled the separation of ligands into functionally distinguishable classes. Importantly, the ligands within each class exhibited similar pharmacological activities, a result that highlights the relationship between receptor structure and activity and provides direction for the discovery of novel AR modulators.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Single-step purification of full-length human androgen receptor.

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 microM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Generation and characterization of human steroidogenic factor 1 LBD crystals with and without bound cofactor peptide.

The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the adrenal gland and gonads, and in sexual differentiation. SF1 performs this pivotal function through the regulation of hormone expression that is essential for organogenesis and endocrine homeostasis. SF1 is a member of a nuclear receptor subclass that contains LRH1 and the Drosophila receptor FTZ-F1. To date, a natural ligand has not been reported for any member of this subfamily. Here we report the crystallization and characterization of the ligand-binding domain (LBD) of human SF1 from two different crystal forms: a binary complex with fortuitous ligand and a ternary complex with the same ligand and a peptide containing a motif of a nuclear receptor cofactor. The structural determination of the binary complex required the use of sulfur SAD phasing, a relatively new technique that uses anomalous diffraction from the endogenous sulfur atoms present in the protein. The structure of the ternary complex was determined by multiple wavelength anomalous diffraction (MAD) using seleno-methionine substituted SF1. Preliminary analysis suggested SF1 contained a fortuitous ligand in the binding pocket. This ligand may account for the relatively high basal activity observed for SF1 in cofactor recruitment and cell-based assays.