Biochemistry and Molecular Biology
Presenter(s): Terrence Lee, Kiersten Chong, Chloe Garcia, Max Strul, Ruchita Kharwa,
Emily Wong, Kevin Bretzing
Advisor(s): Dr. Cedric Owens
Nitrogenase catalyzes the conversion of atmospheric dinitrogen into ammonia. The enzyme produces ammonia under ambient conditions, using the free energy of ATP to drive dinitrogen reduction. The most common form of nitrogenase is molybdenum nitrogenase (Mo-nitrogenase). Mo-nitrogenase is inhibited by the ubiquitous pollutant carbon monoxide (CO). To prevent inhibition of Mo-nitrogenase by CO, nitrogen fixing bacteria produce the protein CowN. In presence of CowN, Mo-nitrogenase avoids inhibition by CO and remains active. However, the mechanism by which CowN protects Mo-nitrogenase is unknown. Enzymatic assays suggest that CowN and Mo-nitrogenase interact with a Kd of approximately 5-10 µM. Here, we present data from crosslinking and pulldown assays that were used to determine how CowN interacts with Mo-nitrogenase. Crosslinking assays using the heterobifunctional crosslinker EDC showed no binding between CowN and Mo-nitrogenase in presence or absence of CO gas. Pulldown assays using Ni-NTA resin with a His-tagged CowN bait also did not show significant binding between CowN to Mo-nitrogenase. These assays indicate that CowN may not have a strong binding enough affinity to Mo-nitrogenase to be detected by crosslinking or pulldown assays. Future work will investigate potential nitrogenase-CowN interaction using fluorescence anisotropy.
Exploring The Effect Of The Diarylpentanoid ca27 On The Degradation Of The Androgen Receptor In Prostate Cancer Cells
Presenter(s): Emma Beale
Advisor(s): Dr. Marco Bisoffi
The present study addresses a possible mechanism of action for the diarylpentanoid curcumin analog 27 (ca27), which has been shown to downregulate the androgen receptor (AR) in prostate cancer (PCa) cells. Prostate cells, both normal and cancerous, express the AR, which functions as a hormone-induced cytoplasmic/nuclear receptor and transcription factor promoting cell growth and survival. In PCa, AR expression and activity are overexpressed, and the AR is a major oncoprotein leading to uncontrolled cell growth. ca27 is a synthetic diarylpentanoid analog of the natural product curcumin. Previous research in Dr. Bisoffi’s lab has shown that ca27 downregulates AR expression at low micromolar concentrations in prostate cancer cells. However, the mechanism of action is unknown. The goal of the present work is to determine at what step of the central biological dogma ca27 acts to downregulate AR expression. Specifically, we hypothesize that ca27 interferes with AR protein stability by enhancing its degradation. Using specific inhibitors of transcription, translation, and degradation, applied to human androgen-dependent LNCaP prostate adenocarcinoma cells, preliminary data is presented based on the method of sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) followed by immunological detection by Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our preliminary findings indicate that ca27 is a mediator of protein degradation. A better understanding of the mechanism of action of ca27 with respect to its activity to downregulate AR expression will build the foundation for the development of organic small molecules as therapeutic candidates in the clinical management of PCa.
Characterizing the Ganglioside Content in Human Intestinal Epithelial Cells and Extracellular Vesicles With or Without Supply of Exogenous Gangliosides
Presenter(s): Jordan Jernigan
Advisor(s): Dr. John Miklavcic
Dietary fats play a role in the development, progression, and treatment of chronic diseases. Gangliosides are a type of lipid, or fat, found in all human tissues. Both healthy and diseased cells undergo intercellular communication by secreting vesicles into the extracellular environment. Extracellular vesicles (EV) contain many bioactive compounds, including gangliosides. Dietary gangliosides are not considered essential nutrients and their health benefits and therapeutic traits remain unidentified. The purpose of this research is to characterize the change in total ganglioside content in human intestinal epithelial cells and EVs due to a treatment with dietary gangliosides. Caco-2 human intestinal epithelial cells were given with an exogenous source of gangliosides (10 μg/mL of GM3 ganglioside or 10 μg/mL of GD3 ganglioside). Cell media was collected at 6h, 24h, and 48h, and EV and ganglioside isolation was complete. EV verification was achieved using immunobotting and electron microscopy. A University of Alberta collaborator will complete lipidomic profiling on cells and EVs. It is anticipated that an exogenous source of gangliosides will alter the total ganglioside content and composition in cells and EVs. The findings of this study will support future research on the therapeutic applications of dietary gangliosides, as well as the physiochemical properties of dietary fats and EVs in relation to human chronic conditions.