Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-hour period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0

Disc-shaped fossils resembling porpitids or eldonids from the early Cambrian (Series 2: Stage 4) of western USA

The morphology and affinities of newly discovered disc-shaped, soft-bodied fossils from the early Cambrian (Series 2: Stage 4, Dyeran) Carrara Formation are discussed. These specimens show some similarity to the Ordovician Discophyllum Hall, 1847; traditionally this taxon had been treated as a fossil porpitid. However, recently it has instead been referred to as another clade, the eldonids, which includes the enigmatic Eldonia Walcott, 1911 that was originally described from the Cambrian Burgess Shale. The status of various Proterozoic and Phanerozoic taxa previously referred to porpitids and eldonids is also briefly considered. To help ascertain that the specimens were not dubio- or pseudofossils, elemental mapping using energy dispersive X-ray spectroscopy (EDS) was conducted. This, in conjunction with the morphology of the specimens, indicated that the fossils were not hematite, iron sulfide, pyrolusite, or other abiologic mineral precipitates. Instead, their status as biologic structures and thus actual fossils is supported. Enrichment in the element carbon, and also possibly to some extent the elements magnesium and iron, seems to be playing some role in the preservation process

Development of a Grp94 inhibitor

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja303477g.Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein

Physical characterixation and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size enriched into different size bins by low speed centrifugation or a combination of gravitational sedimentation and Fluorescence-Activated Cell Sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5–10 μm in size displayed elevated cytokine release profiles compared to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by Microflow imaging, Transmission Electron Microscopy, and Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in-vitro PBMC studies to rank order the immunogenic potential of various types of mAb particles is discussed

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore-forming toxins

This research was originally published in the Journal of Biological Chemistry. Suman Ghosh, Heather E. Shinogle, Nadezhda A. Galeva, Rick T. Dobrowsky, and Brian S. J. Blagg. Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport. Journal of Biological Chemistry. 2016; 291, 8309-8323.Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion

Antigen-specific blocking of immunological synapse formation using bifunctional peptide, Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes

This is the publisher's version.The long-term objective of this project is to utilize the I-domain protein for the alpha-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results in-dicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes

Hsp90 C‑Terminal Inhibitors Exhibit Antimigratory Activity by Disrupting the Hsp90α/Aha1 Complex in PC3-MM2 Cells

Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure–function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors