Pyruvate kinase M2 (PKM2) has been studied for decades on its role in cancer metabolism. Recently, PKM2 is highlighted again for its new function: promoting gene transcription by acting as a protein kinase. Moreover, the PKM2 levels in patient circulation have been used as a diagnostic marker for many types of cancers. However, it remains unclear whether PKM2 in blood circulation has any physiological or pathological function. In my dissertation, I demonstrate that PKM2 released from cancer cells facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes endothelial cell proliferation, migration and survival. Only the dimeric PKM2, not the tetrameric PKM2 possesses the activity in angiogenesis promotion. Our results further indicate that PKM2 regulates angiogenesis by integrin αvβ3 activation and integrin redistribution. I also found that PKM2 enhances drug resistance of cancer cells expressing integrin αvβ3.

Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 101-118).

There is a growing body of interest to understand the regulation of allosteric proteins. Allostery is a phenomenon of protein regulation whereby binding of an effector molecule at a remote site affects binding and activity at the protein‟s active site. Over the years, these sites have become popular drug targets as they provide advantages in terms of selectivity and saturability. Both experimental and computational methods are being used to study and identify allosteric sites. Although experimental methods provide us with detailed structures and have been relatively successful in identifying these sites, they are subject to time and cost limitations. In the present dissertation, Molecular Dynamics Simulations (MDS) and Principal Component Analysis (PCA) have been employed to enhance our understanding ofallostery and protein dynamics. MD simulations generated trajectories which were then qualitatively assessed using PCA. Both of these techniques were applied to two important trypanosomatid drug targets and controlling enzymes of the glycolytic pathway - pyruvate kinase (PYK) and phosphofructokinase (PFK). Molecular Dynamics simulations were first carried out on both the effector bound and unbound forms of the proteins. This provided a framework for direct comparison and inspection of the conformational changes at the atomic level. Following MD simulations, PCA was run to further analyse the motions. The principal components thus captured are in quantitative agreement with the previously published experimental data which increased our confidence in the reliability of our simulations. Also, the binding of FBP affects the allosteric mechanism of PYK in a very interesting way. The inspection of the vibrational modes reveals interesting patterns in the movement of the subunits which differ from the conventional symmetrical pattern. Also, lowering of B-factors on effector binding provides evidence that the effector is not only locking the R-state but is also acting as a general heat-sink to cool down the whole tetramer. This observation suggests that protein rigidity and intrinsic heat capacity are important factors in stabilizing allosteric proteins. Thus, this work also provides new and promising insights into the classical Monod-Wyman-Changeux model of allostery.

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Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle. / Master of Science

Ethyl pyruvate, a known ROS scavenger and anti-inflammatory drug was found to combat leukemia cells. Tumor cell killing was achieved by concerted action of necrosis/apoptosis induction, ATP depletion, and inhibition of glycolytic and para-glycolytic enzymes. Ethyl lactate was less harmful to leukemia cells but was found to arrest cell cycle in the G0/G1 phase. Both, ethyl pyruvate and ethyl lactate were identified as new inhibitors of GSK-3β. Despite the strong effect of ethyl pyruvate on leukemia cells, human cognate blood cells were only marginally affected. The data were compiled by immune blotting, flow cytometry, enzyme activity assay and gene array analysis. Our results inform new mechanisms of ethyl pyruvate-induced cell death, offering thereby a new treatment regime with a high therapeutic window for leukemic tumors.