My name is Rob Parry, and I began my summer research a few weeks ago in Dr. Ian Blair’s Pharmacology Lab at the University of Pennsylvania. The lab studies metabolism using mass spectroscopy techniques. Mass spectroscopy is a fancy technique that detects molecules based on their mass to charge ratio by ionizing the sample (giving it a charge), exposing it to a uniform magnetic field, and detecting how much the magnetic field deflects the molecules flight path. With this information, the mass spectrometer calculates the mass to charge ratio of the molecules it detects. Mass spectroscopy is a sensitive method to detect various metabolites and can accurately quantify how much of each metabolite there is. My main project is to study the effects of inhibiting Coenzyme A (CoA) biosynthesis utilizing mass spectroscopy techniques.
CoA is a molecule common to most, if not all, organisms and generally acts as an acyl-group carrier. CoA forms high energy thioester bonds with metabolites in a number of metabolic pathways including but not limited to the Krebs Cycle, fatty acid biosynthesis, fatty acid beta-oxidation, and isoprenoid biosynthesis. It is estimated that CoA and its thioester derivatives take part in 4% of all cellular reactions in E. Coli (Wow!).
Pantothenate Kinase (PanK) is an enzyme crucial in CoA biosynthesis: it catalyzes the phosphorylation of pantothenic acid in the rate limiting step of CoA biosynthesis. Genetic defects in PanK can result in neurodegeneration. In addition, preliminary studies have shown that rotenone, a toxic molecule with a known association to Parkinson’s Disease, also impairs CoA biosynthesis. Simply put, studying the inhibition of CoA biosynthesis may lead to insight on how neurodegenerative diseases come about.
In order to inhibit CoA biosynthesis, we are using hopantenic acid, a previously described competitive inhibitor of PanK. Initial experiments were to confirm that hopantenic acid inhibitis CoA biosynthesis.
Isotopic labelling studies of pantothenic acid, a precursor of CoA and the natural substrate of PanK, were conducted. If hopantenic acid affected CoA biosynthesis, we should have seen decreased incorporation of isotopically labelled pantothenic acid in Coenzyme A in cells exposed to hopantenic acid. After 2 attempts, we saw did not see diminished labelling due to the presence of hopantenic acid (bummer). Studies with longer incubation time and higher concentrations of the inhibitor in fresh media enriched with isotopically labelled pantothenic acid will be conducted next. However, a few mishaps in cell culturing have caused delays in that project.
In the meantime, one of the other project we have been working on is “LC-MS analysis of human platelets as a platform for studying mitochondrial metabolism” for the Journal of Visualized Experiments. The lab has previously used platelets as a platform to study the metabolic effects of various drugs. Platelets are useful as they can be easily obtained in a large quantity from donors or from blood blanks. While cancer cell lines are often used to study metabolic effects of various drugs, cancer cells metabolize differently than normal cells. In addition, as platelets lack a nucleus, metabolic alterations in platelets are independent of transcriptional regulation, which can be useful to specifically study mitochondrial metabolism. For these reasons, platelets can offer a useful platform for studying mitochondrial metabolism.
I am enjoying my time here and I am learning a lot both about metabolism and mass spectroscopy as a technique. The people in the lab are all nice and very knowledgeable. I get to run cool experiments, and, sometimes, I even get to play with dry ice.