Todd Pray

Differentiation-dependent expression of E1^E4 proteins in cell lines maintaining episomes of human papillomavirus type 31b

The life cycle of human papillomaviruses (HPVs) is dependent on epithelial differentiation. Among the viral proteins expressed in differentiated epithelial cells are the viral capsid proteins, L1 and L2, as well as the E1E4 fusion proteins. In this study, the expression and intracellular localization of the E1E4 proteins of HPV type 31b were examined in both monolayer and raft cultures of the CIN-612 cell line which maintains episomal copies of HPV-31b. In this cell line, a high level of E1E4 protein expression was observed in the cytoplasm of a small percentage of cells in monolayer culture. A large increase in E1E4 protein levels was observed upon stratification of the CIN-612 cell line in raft cultures, with E1E4 protein expression limited to the uppermost layers of the epithelium. A diffuse, slightly grainy cytoplasmic localization of E1E4 protein was observed in both monolayer and raft culture systems. Although virion synthesis is entirely dependent upon phorbol ester or synthetic diacylglycerol treatment of raft cultures, E1E4 expression was observed in both treated and untreated monolayer and raft cultures of the CIN-612 cell line. In monolayer cultures of two simian virus 40-transformed cell lines, cos-7 and MK-6, transiently transfected with an E1E4 expression vector, the distribution of E1E4 protein was found to differ substantially from that in the CIN-612 cells. In these cell lines E1E4 protein was found to exhibit a total collapse into either cytoplasmic inclusion granules in the cos-7 cells or a perinuclear halo-like structure in the MK-6 cell line. The host cell, its differentiation state, and the amount of expression can therefore significantly affect the distribution of the E1E4 proteins.

Discovery of ADDL-Targeting Small Molecule Drugs for Alzheimers Disease

Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimers disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.

A Homogeneous FRET Assay System for Multiubiquitin Chain Assembly and Disassembly

Ubiquitin (Ub, 76aa) is a small highly conserved protein present universally in eukaryotic cells. Covalent attachment of (Ub)(n) to target proteins is a well-known posttranslational modification that has been implicated in a wide array of cellular processes including cell biogenesis. Ubiquitin polymerization by the Ub activation-conjugation-ligation cascade and the reverse disassembly process catalyzed by Ub isopeptidases largely regulate substrate protein targeting to the 26S proteasome. Ub chains of four or more subunits attached by K48 isopeptide linkages have been shown to be necessary for the 26S proteasome association and subsequent degradation of protein molecules. To better understand this protein degradation event, it is important to develop Ub polymerization and depolymerization assays that monitor every reaction step involved in Ub attachment to, or detachment from, substrate protein molecules. In this chapter, we describe homogeneous, easy-to-use, nonradioactive, complementary continuous fluorescence assays capable of monitoring the kinetics of Ub chain formation by E3 Ub ligases, and their hydrolysis by isopeptidases, which rely on mixing a 1:1 population of fluorophore-labeled Ub molecules containing a FRET pair. The proximity of fluorescein (donor) and tetramethylrhodamine (acceptor) in Ub polymers results in fluorescein quenching on ligase-induced Ub chain assembly. Conversely, a dramatic enhancement of fluorescein emission was observed on Ub chain disassembly because of isopeptidase activity. These assays thus provide a valuable tool for monitoring Ub ligase and isopeptidase activities using authentic Ub monomers and polymers as substrates. Screening of a large number of small molecule compound libraries in a high-throughput fashion is achievable, warranting further optimization of these assays.