Andrew McGowan

INTRODUCTION

Everyday, we endure physiological stressors that can damage our proteins

and cells. Things like cold or hot temperatures, exercise, UV light, and alcohol can

cause proteins to misfold and clump together. This misfolding and aggregating

process plays a role in the formation of plaque in the brain and the solid yolk in

eggs. Fortunately, most organisms possess a cellular defense mechanism called the

heat shock response (HSR), which either helps these proteins refold into their usual

shape or initiates their degradation. The HSR plays a role in regulatory and

inflammatory processes and contributes to the resilience of several types of cancer

cells. We also know that small electrophilic molecules can directly upregulate the

HSR; however, the exact mechanism of activation is not fully understood.

This past year, I worked in Prof. Rebecca Connor’s lab, investigating how

parthenolide-- an electrophile commonly found in daises-- interacts with the heat

shock response in acute myeloid leukemia cells (THP-1). Previous research found

that parthenolide selectively eradicates leukemia cells and upregulates the HSR

through binding to heat shock proteins involved in the heat shock repsonse.

METHODS & RESULTS

        My main project over the summer was to isolate and identify several key proteins from cells treated with an alkyne-bearing parthenolide derivative. To do this, we reacted the treated cells with azide-tagged biotin, affinity purified all biotinylated proteins using avidin, and identified modified proteins via western blotting. We also treated purified Hsp70 with parthenolide and aimed to identify the specific sites of modification using MALDI-TOF/TOF mass spectrometry. Previous results from in vitro experiments indicate that parthenolide adducts with heat shock proteins 70 and 90 (Hsp70; Hsp90), triggering the production of even more heat shock proteins.

        We were eventually able to isolate Hsp70 from treated THP-1 cells, however we could not see any sign of modification from the mass spectrophotometry data, most likely because parthenolide-bound Hsp70 peptides are difficult to identify, due to parthenolide’s non-polar nature. Future experiments will employ a peptidomic approach to identify the sites of modification on Hsp70, replicate my results, and determine whether, parthenolide adducts with Hsp40 and/or Heat Shock Transcription Factor 1 as well.

At the end of the summer, I presented these findings at the American

Chemical Society’s national conference.

For more information about this project,

contact Professor Rebecca Connor at connorr@dickinson.edu