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2 results on '"Peter R. Strack"'

1. Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression.

Author

Peter J Stivers, Lauren Harmonay, Alexandra Hicks, Huseyin Mehmet, Melody Morris, Gain M Robinson, Peter R Strack, Mary J Savage, Dennis M Zaller, Izabela Zwierzynski, and Philip E Brandish

Subjects
Medicine, Science
Abstract

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid-induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

Published
2015
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2. Activation of the Multicatalytic Endopeptidase by Oxidants. Effects on Enzyme Structure

Author

Lloyd Waxman, Julie M. Fagan, and Peter R. Strack

Subjects
Male, Proteasome Endopeptidase Complex, Proteases, Erythrocytes, Serine Proteinase Inhibitors, Protein Conformation, Iron, Proteolysis, medicine.medical_treatment, Molecular Sequence Data, p-Chloromercuribenzoic Acid, Biochemistry, Enzyme activator, Coumarins, Multienzyme Complexes, medicine, Animals, Amino Acid Sequence, Enzyme Inhibitors, Serine protease, Protease, medicine.diagnostic_test, biology, Chemistry, Hydrolysis, Proteins, Hydrogen Peroxide, Oxidants, Cysteine protease, Endopeptidase, Enzyme structure, Enzyme Activation, Cysteine Endopeptidases, Isocoumarins, biology.protein, Peptides, Chickens, Chloromercuribenzoates
Abstract

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.

Published
1996
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