Investigating glycolysis regulation: establishing molecular tools to manipulate glycolytic biosynthesis
Denis Titov, Assistant Adjunct Professor
Nutritional Sciences and Toxicology
Closed. This professor is continuing with Fall 2023 apprentices on this project; no new apprentices needed for Spring 2024.
Glycolysis is the focal point of mammalian metabolism - not only for its ability to efficiently generate energy, but also its capacity to support biosynthesis networks. Catabolizing glucose through a series of highly conserved enzymatic reactions, glycolysis yields precursors and cofactors that contribute to anabolic pathways like lipid and amino acid synthesis. The direction and magnitude of glycolytic flux is highly regulated through a combination of hormonal signaling and allosteric effectors directly binding to and modifying enzymatic activity. Fructose-2,6-bisphosphate (F26BP) is the most potent allosteric activator of the glycolysis rate-limiting enzyme 6-phospho-1-fructokinase (PFK1), and therefore a capable of enhancing glycolytic flux even in under inhibitory conditions. However, the function of F26BP in cell metabolism is unclear as its allosteric action fully redundant with other glycolysis regulators like ADP and inorganic phosphate. The overarching hypothesis of the project is that the function of F26BP allostery is to override glycolysis regulation by energy charge (i.e. high ATP/ADP ratio) and promote biosynthesis. The immediate goal of the project is to quantitatively define the role of F26BP in cell metabolism by developing: (a) molecular tools to pointedly manipulate F26BP concentrations in live cells and (b) an in vitro assay to characterize metabolic conditions that favor F26BP synthesis.
Role: Over the course of this project, students will gain proficiency with the development of molecular tools used to examine cellular metabolism and aspire for investigative independence. Specific techniques will include: (1) polymerase chain reaction (PCR) and subsequent cloning techniques, (2) recombinant protein purification, and (3) gel electrophoresis analyses like Western Blotting and Coomassie Staining. Upon greater involvement with the project, there will be additional opportunities to gain experience with tissue culture techniques, and analyzing Seahorse (quantifies glycolytic vs. respiratory capacity in live cells) and mass spectrometry (quantifies metabolite concentrations) data. Students will also gain greater exposure to reading primary research papers and facilitating scientific presentations.
If interest and productivity persist throughout the semester, there will be opportunities to continue this project in future semesters. With greater project involvement, independent research can as the foundation for an honors thesis. All contributions to the advancement of the project will lead to inclusion as an author on future presentations and publications.
Qualifications: Undergraduates seeking to apply should be: (1) highly motivated, organized and engaged, (2) clear communicators (esp. with scheduling & experimental issues), (3) perseverant, and (4) eager to ask questions and learn from mistakes. This position is recommended for those interested in molecular biology and biochemistry, especially as it pertains to cellular metabolism. Previous research experience (although valuable) is not required, but an enthusiasm to learn is absolutely essential. Ideally, the apprentice will have some previous, conceptual exposure to molecular biology and cellular metabolism. Highly interested in accepting students of current Sophomore or Junior standing due to the possibility of continuing research in the future, but a current Senior with sufficient previous experience can also be considered.
Day-to-day supervisor for this project: Megan Kober, Graduate Student
Hours: 12 or more hours
Related website: http://denistitovlab.org
Biological & Health Sciences