Activation of Enzymes

1. Activation of Enzymes, HIV, and HSPs NSF Grant # DUE0653234 David Coreas Molecular Biology dvdcoreas@yahoo.com Elena Betancourt Physiology and Neuroscience ebetancourt@ucsd.edu Melody Sycks Biochemistry msycks@ucsd.edu Introduction Method Results Conclusion Activation of Enzymes The goal of Moores UCSD Cancer Center's Continuing Umbrella of Research Experiences program in Cancer Cells, is to understand the glucose metabolism in cancer tissues by measuring the activity of enzymes: 1phosphofructokinase (PFK-1), Muscle 1phosphofructokinase (mPFK1), Liver 1phosphofructokinase (lPFK1) heart 1phosphofructokinase (hPFK1), lactate dehydrogenase (mLDH). The role/effect that Vitamin C has in glycolysis. PFK-1  Hsp’s The aggregation (clumping together of denatured proteins) of proteins is harmful to cells, and are indicatory of diseases such as Alzheimer's, Parkinson’s, viral disease, and fever. Hep G2 (Hepatocellular Carcinoma), produce different heat shock proteins (HSP’s), as a defense mechanism against heat stress. Hsp70 prevents protein aggregation; Hsp90 is responsible for the creation, destruction, and maintenance of proteins; both HSP70 and HSP90 assist in the folding of proteins; HSP27 acts as a chaperone to preventing denatured proteins from aggregating and plays an important role in stabilizing them. HSP90  HIV Three decades of Human Immunodeficiency Virus (HIV) research has resulted in various antiviral drugs which target viral proteins such as Protease (PR), Reverse Transcriptase, Integrase, or the cellular receptors involved in viral entry. Roughly 32 inhibitors, 11 of which target protease, have been approved by the FDA since the first Protease Inhibitor (PI) was launched in 1995. With the use of Green Fluorescent Proteins (GFP), acting as a downstream reporter, assays can be developed that show whether or not Protease has been inhibited. HIV Virus  Activation of Enzymes Hsp’s Protein concentration assay, LDS-PAGE, PCR and Western Blot techniques were used to locate and identify heat shock proteins. Hsp70 and Hsp27 were induced by heat shock, whereas the presence of Hsp90 was independent of heat shock. This means that Hsp70 and Hsp27 production in a cell increases when exposed to heat stress. Heat stress is not a factor in Hsp90 production. Activation of Enzymes To measure the activity of the enzymes, different methods were used: Dimensional analysis (mathematical calculation) to weigh chemicals and prepare dilutions, spectrophotometers used to measure inhibition/purification/activity/stabilization of muscle enzymes (phosphofructokinase-1, PFK-1; aldolase; and lactate dehydrogenase ,mLDH) Table 1: 50 grams of rabbit muscle measured in different concentrations and environments at volumes from 150mls to 220mls. Hsp’s The experiment being conducted on Hsp70, 90, and 27, involves locating and identifying Hsp’s. Data is then analyzed to determine their relative concentrations after predetermined stress conditioning. The induction of Hsp’s in HepG2 is measured through the use of Protein Concentration Assay, LDS-PAGE, PCR, and Western Blot techniques. Interpretation: -Hsp24 production progressively increased with resting time after stress. (30ug/µl) (10 ug/µl) Graph 1: Represents an equilibrium of enzymes from 200mls to 220mls. HIV Research on peptide libraries acting as Protease Inhibitors is ongoing novel research , and any conclusions drawn at the moment are speculative. Activation of Enzymes From 50 grams of rabbit muscle, there were 40.3 total enzymes units, a 4.26% yield of enzymes, a specific activity of 0.61 eu/mg, and a 5.2 fold purification. HIV Research is ongoing. Hsp’s Illustrates the process of DNA amplification HIV The premise of the assay is to monitor the effectiveness of a protease inhibitor by the use of five major components: Protease (viral-enzyme), Protease Inhibitor (peptide library), Upstream Activation Sequence (‘UAS’/promoter sight), GFP (Downstream Reporter), and Gal4 (2-domain structure that binds to the UAS; if un-cleaved by viral-enzymes). By expressing a random peptide library within the cytoplasm of a cell, it is possible to interfere with the proteolytic activity of protease and keep it from cleaving the long precursor protein of HIV into the functional HIV proteins. Western Blot Results Hsp90 Low Concentration -----(10ug/20µl) High concentration (30 ug/20µl )----> Acknowledgements Hep G cells were exposed to heat stress and then rested for different periods of time. Four different examples are shown: Standard “C,” No rest time “HS0,” four hours rest “Hs4,” and twenty-four hours rest “Hs24.” Interpretation: -Pictured above: results from Hsp90 show no change in concentration. -Picture blow: results from Hsp70 show progressive increase in concentration over resting times. Special thanks to: Moores Cancer Center, National Science Foundation, LIPP Family Foundation, M.E.S.A. Program , Wolkowicz Laboratory, De Maio Laboratory, Percy Russell Laboratory, Dr. Lawrence Alfred, Dr. Raga Bakhiet, Dr. Nouna Bakhiet, Dr. Antonio De Maio, Dr. David Cauvi, Dennis Hawisher B.S., Dr. Percy Russell, Dr. Roland Wolkowicz, Aleksandr Stotland PhD Candidate, Dean Kathy Tyner, and Dr. Percy Russell Western Blot Results Hsp70 High concentration (30 ug/20µl) Low Concentration (10ug/20µl) Assay developed to monitor proteolytic activity by expression of green-florescent protein. (HS0, HS24, HS4, C)