362091 Quantification of Deubiquitinating Enzyme Activity in Cancer Cells Using a Protease-Resistant, Peptide-Based Reporter

Monday, November 17, 2014: 12:30 PM
205 (Hilton Atlanta)
Adam Melvin, Chemical Engineering, Louisiana State University, Baton Rouge, LA

The success of chemotherapeutics depends on the selective, efficient targeting of enzymes and proteins known to be deregulated in specific cancers. Unfortunately, there currently exists a critical bottleneck associated with inadequate reporting technology to properly assess enzyme activity and drug effectiveness. One such enzymatic class, deubiquitinating enzymes (DUBs), are essential enzymes that regulate protein degradation, cell division, and cell death. Hyper-regulation of DUBs has recently been linked to increased drug resistance and tumor growth in numerous cancers including multiple myeloma, leukemia, and colorectal cancer. While current technologies attempt to recapitulate DUB activity in vitro, it has become clear that cell-free, test-tube based reactions do not adequately mimic in vivo conditions, resulting in the high failure rate of DUB-based therapeutics. To more closely reconstitute the clinical environment, reporting schemes must be developed that are capable of measuring enzyme activity in intact, single cells in a high-throughput manner to account for the heterogeneous nature of cancer cells and tumors.

This presentation will highlight recent work on a fluorescent, peptide-based reporter that is specifically designed to be (1) easily incorporated into intact cells, (2) resistant to degradation by intracellular protease, (3) specific to DUBs, and (4) compatible with single cell analysis techniques like droplet microfluidic devices. Peptide-based reporters have demonstrated remarkable success as biosensors for proteins relevant to cancer due to their ability to be directly incorporated into intact cells without the need for genetic engineering. A key component of the new DUB reporter is the incorporation of a β-hairpin ‘protectide’ at the N-terminus of a common DUB-binding substrate to prevent rapid intracellular degradation.  These protectides have been demonstrated to confer significant stability to peptide-based reporters.  Further, we show how the protectide also serves as a cell penetrating peptide (CPP) to facilitate entry into intact cells.  Reporter performance was assessed by measuring the increase in fluorescence due to DUB-mediated cleavage of a C-terminal fluorophore.  Reporter stability was evaluated using a standard degradation assay and reverse phase high performance liquid chromatography (RP-HPLC). Cellular uptake of the reporter was determined by confocal microscopy and quantified by fluorescence emission using 96-well plates. At each step, the performance of the peptide-based reporter was compared to several commercially available DUB reporting schemes.  The work outlined in this presentation highlights the first step in a novel reporting scheme to assess DUB activity in single cancer cells across a highly heterogeneous environment, like a patient biopsy.

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