Pyruvate kinase (PK)在醣解作用(Glycolysis)中扮演重要的角色，其可轉移Phosphoenolpyruvic acid (PEP)的磷酸根至Adenosine diphosphate (ADP)以產生丙酮酸(pyruvate)。其中丙酮酸更可進一步合成acetyl-coA以進入Tricarboxylic acid cycle (TCA cycle)產生更多的能量，這種能量的生成方式表現在正常細胞中。然而在癌細胞中Pyruvate kinase muscle 2 (PKM2)被大量的表現，此蛋白在Warburg effect及有氧糖解作用(Aerobic glycolysis)中扮演重要的角色。即使在充足氧氣的環境，PKM產生Pyruvate依然藉由Lactate dehydrogenase 催化生成Lactate。然而PKM2是一種活性較差的激酶，此特性一般認為是為了累積糖解作用中的代謝物以利細胞快速生長。此外，PKM2 可以進入細胞核中，和包含Hypoxia-inducible factor 1 (HIF-1)等轉錄因子成為轉錄作用的活化物，增加癌症相關的代謝。H391Y 和 K422R是一種自然發生的基因突變，會使得第391個胺基酸從Histidine 轉變為Tyrosine，以及第422個胺基酸從Lysine轉變為Arginine，這兩種突變發生於布隆氏症候群 (先天性毛細血管擴張性紅斑)的患者身上，且此種遺傳疾病也伴隨易產生癌症的表徵。PKM2的結構型態會隨著與調控物的結合改變，例如，與Fructose_1,6-bisphosphate (FBP)結合即形成高活性的R(relax)-stat。本研究解出H391Y的晶體結構，其和Glutamate 386形成氫鍵，加入Phenylalanine時可防止Alpha-helix (residues 371-388)位移，使得H391Y 處於高活性、類似 R-state的型態；相反的，人為突變的R399E為第399個胺基酸從Arginine轉變為Glutamate，相較於WT 的Arg 399 與另一domain的Glu 418 形成四個氫鍵，突變株中Glu 399另一domain的Glu418的鍵結只有兩個氫鍵，所以弱化R399E C-domain 之間的連結作用，並使R399E 產生oligermeric的型態變化 (tetramer 與 dimer 之平衡)，dimer結構的PKM2 能進入細胞核內磷酸化transcription activator促進腫瘤的增生。

Abstract
Pyruvate kinase (PK), a crucial enzyme in glycolysis, catalyzes the transfer of phosphate from Phosphoenolpyruvic acid (PEP) to Adenosine diphosphate (ADP) to produce pyruvate, which is further synthesized into acetyl-coA and enters into Tricarboxylic acid cycle (TCA cycle) for high-energy production in normal cells. In cancer cell, an isoform Pyruvate kinase muscle 2 (PKM2) is frequently overexpressed; it is now recognized that PKM2 is a key molecule involved in Warburg effect, or aerobic glycolysis. Even in the presence of sufficient oxygen, the product pyruvate primarily shifts to the production of lactic acid by lactate dehydrogenase. PKM2 is a less efficient isoform of pyruvate kinase which is thought to lead to the accumulation of glycolytic intermediates for rapid cell growth. Structures of PKM2 show conformational change with or without that the allosteric effector fructose 1, 6-biphosphate (FBP), in which the FBP-binding complex stays at an active R-state conformer. H391Y and K422R are natural variants found from Bloom syndrome patients, prone to developing cancer. R399E, an artificial mutant is also reported to lead to the development of cancer. In this study, we aimed to understand the structure-activity relationship of these variants in allosterically modulate enzymatic activity. We have determined H391Y and R399E structure, showing an overall R-state-like conformation. Notably, Tyr 391 could strongly hydrogen-bond with Glu 386 which could prevent alpha-helix translation in opposition to WT with phenylalanine. R399E stays in loose oligomeric conformation, as a result of fewer contacts between Glu 399 with the neighbor subunit (Glu 399 makes 2 H bonds with Glu 418 in the neighbor subunit as opposed to 4 H bonds between Arg 399 and Glu 418 in the WT enzyme). The lower allosteric effect by FBP and serine seen in R399E supports the contribution of oligomeric interactions to allosteric modulation. Our results demonstrate that PKM2 variants H391Y and R399E affects the modulation of allosteric effect and oligomeric interactions, which may further contribute to disease development.

Index
中文摘要 1
Abstract 2
誌謝 3
List of Tables 6
List of Figures 7
List of Abbreviation 8
1. Introduction 9
1.1 Pyruvate kinase 9
1.2 PKM2 is involved in Warburg effect 10
1.3 Allosteric regulation of PKM2 in the cancer metabolism 10
1.4 Post-translational modification of PKM2 12
1.5 Nucleus translocation 13
1.6 Protein kinase activity of PKM2 13
1.7 Therapeutic targets 14
1.8 H391Y and K422R missense mutation 15
2. Material and methods 16
2.1 PKM2 protein expression 16
2.2 Protein purification 16
2.3 Protein condensation and dialysis 17
2.4 Determination of protein concentration 17
2.5 Lactate Dehydrogenase (LDH) – coupled pyruvate kinase assay 17
2.6 Modification of PKM2 crystallization condition 18
2.7 X-ray diffraction and data collection 18
2.8 PKM2 structures building and refinement 19
2.9 Structure comparison and analysis 19
3. Results 21
3.1 Protein expression and purification of wild-type and PKM2 variants 21
3.2 Enzymatic characterization of PKM2 vs. PKM2 variants 21
3.2.1 Mechanism of transphosphorylase and LDH-coupled assay 21
3.2.2 Pyruvate standard curve 21
3.2.3 Enzymatic features (Km, kcat) of WT PKM2 and activity comparison among serine, FBP, and phenylalanine 21
3.2.4 Enzymatic features (Km, kcat) of H391Y and activity comparison among serine, FBP, and phenylalanine 22
3.2.5 Enzymatic features (Km, kcat) of R399E and activity comparison among serine, FBP, and phenylalanine 23
3.3 Crystallization, data collection, and structure refinement of PKM2 variants 23
3.3.1 Structure of PKM2 variants 23
3.3.2 Crystallization of H391Y and R399E mutant PKM2 23
3.3.3 X-ray diffraction data collection and process 24
3.3.4 Structure model building, refinement, and validation 24
3.3.5 Identification of mutant site with the density map 24
4. Discussion 25
4.1 Allosteric regulator change from serine, FBP, and phenylalanine 25
4.1.1 Serine raises wild-type PKM2 to higher activity 25
4.1.2 Phenylalanine changes wild-type PKM2 from normal-state to T-state 25
4.1.3 FBP regulates B domain and Arg 342 beside catalytic site 25
4.2 Allosteric regulation from PKM2 variants 26
4.2.1 H391Y mutant PKM2 behaves like FBP in assay and remains rigider conformation 26
4.2.2 R399E mutant PKM2 leads tetrameric conformation change and lower activity 27
4.2.3 Allosteric regulation among R399E, FBP, and phenylalanine 27
4.3 Different allosteric behavior of wild-type PKM2 and PKM2 variants has its biological function 28
5. References 30

1. Noguchi, T., H. Inoue, and T. Tanaka, The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. J Biol chem, 1986. 261(29): p. 13807-12.
2. Noguchi, T., et al., The L-Type and R-Type Isozymes of Rat Pyruvate-Kinase Are Produced from a Single Gene by Use of Different Promoters. J Biol chem, 1987. 262(29): p. 14366-14371.
3. Carbonell, J., et al., Pyruvate kinase. Classes of regulatory isoenzymes in mammalian tissues. Eur J Biochem, 1973. 37(1): p. 148-56.
4. Yamada, K. and T. Noguchi, Regulation of pyruvate kinase M gene expression. Biochem Biophys Res Commun, 1999. 256(2): p. 257-62.
5. Brinck, U., et al., L-Pyruvate and M(2)-Pyruvate Kinase Expression in Renal-Cell Carcinomas and Their Metastases. Virchows Archiv-an International Journal of Pathology, 1994. 424(2): p. 177-185.
6. Domingo, M., et al., Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies. J Histochem Cytochem, 1992. 40(5): p. 665-73.
7. Rodriguezhorche, P., et al., Comparative Kinetic-Behavior and Regulation by Fructose-1,6-Bisphosphate and Atp of Pyruvate-Kinase from Erythrocytes, Reticulocytes and Bone-Marrow Cel. Comp Biochem Physiol, Part B: Biochem Mol Biol, 1987. 87(3): p. 553-557.
8. Yamada, K. and T. Noguchi, Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochem J, 1999. 337 ( Pt 1): p. 1-11.
9. Reinacher, M. and E. Eigenbrodt, Immunohistological demonstration of the same type of pyruvate kinase isoenzyme (M2-Pk) in tumors of chicken and rat. Virchows Arch B Cell Pathol Incl Mol Pathol, 1981. 37(1): p. 79-88.
10. Hacker, H.J., P. Steinberg, and P. Bannasch, Pyruvate kinase isoenzyme shift from L-type to M2-type is a late event in hepatocarcinogenesis induced in rats by a choline-deficient/DL-ethionine-supplemented diet. Carcinogenesis, 1998. 19(1): p. 99-107.
11. Saheki, S., et al., Hybrid Isozymes of Rat Pyruvate-Kinase - Their Subunit Structure and Developmental-Changes in Liver. Biochem Biophys Acta, 1978. 526(1): p. 116-128.
12. Semenza, G.L., Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003. 3(10): p. 721-32.
13. Semenza, G.L., et al., Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol chem, 1994. 269(38): p. 23757-63.
14. Semenza, G.L., HIF-1: upstream and downstream of cancer metabolism. Curr Opin Genet Dev, 2010. 20(1): p. 51-6.
15. Papandreou, I., et al., HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab, 2006. 3(3): p. 187-97.
16. Semenza, G.L., et al., Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol chem, 1996. 271(51): p. 32529-37.
17. Kim, J.W., et al., HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab, 2006. 3(3): p. 177-85.
18. Luo, W. and G.L. Semenza, Pyruvate kinase M2 regulates glucose metabolism by functioning as a coactivator for hypoxia-inducible factor 1 in cancer cells. Oncotarget, 2011. 2(7): p. 551-6.
19. Warburg, O., On the origin of cancer cells. Science, 1956. 123(3191): p. 309-14.
20. Luo, W.B., et al., Pyruvate Kinase M2 Is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor 1. Cell, 2011. 145(5): p. 732-744.
21. Mazurek, S., Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. Int J Biochem Cell Biol, 2011. 43(7): p. 969-80.
22. Christofk, H.R., et al., The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature, 2008. 452(7184): p. 230-U74.
23. Mazurek, S., et al., Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol, 2005. 15(4): p. 300-8.
24. Ashizawa, K., et al., In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate. Journal of Biological Chemistry, 1991. 266(25): p. 16842-6.
25. Dombrauckas, J.D., B.D. Santarsiero, and A.D. Mesecar, Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry, 2005. 44(27): p. 9417-29.
26. Christofk, H.R., et al., Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature, 2008. 452(7184): p. 181-U27.
27. Chaneton, B., et al., Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature, 2012. 491(7424): p. 458.
28. Williams, R., et al., Differentiating a ligand's chemical requirements for allosteric interactions from those for protein binding. Phenylalanine inhibition of pyruvate kinase. Biochemistry, 2006. 45(17): p. 5421-9.
29. Hitosugi, T., et al., Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal, 2009. 2(97): p. ra73.
30. Anastasiou, D., et al., Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science, 2011. 334(6060): p. 1278-83.
31. Eigenbrodt, E., et al., Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog, 1992. 3(1-2): p. 91-115.
32. Eigenbrodt, E., et al., Structural and Kinetic Differences between the M2-Type Pyruvate Kinases from Lung and Various Tumors. Biomedica Biochimica Acta, 1983. 42(11-1): p. S278-S282.
33. Marchut, E., M. Guminska, and T. Kedryna, The Inhibitory Effect of Various Fatty-Acids on Aerobic Glycolysis in Ehrlich Ascites Tumor-Cells. Acta Biochimica Polonica, 1986. 33(1): p. 7-16.
34. Keller, K.E., I.S. Tan, and Y.S. Lee, SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucose-limited conditions. Science, 2012. 338(6110): p. 1069-72.
35. Chen, N., et al., The oxygen sensor PHD3 limits glycolysis under hypoxia via direct binding to pyruvate kinase. Cell Res, 2011. 21(6): p. 983-6.
36. Spoden, G.A., et al., The SUMO-E3 Ligase PIAS3 Targets Pyruvate Kinase M2. Journal of Cellular Biochemistry, 2009. 107(2): p. 293-302.
37. Zhao, S., et al., Regulation of cellular metabolism by protein lysine acetylation. Science, 2010. 327(5968): p. 1000-4.
38. Lv, L., et al., Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth. Molecular Cell, 2011. 42(6): p. 719-730.
39. Hoshino, A., J.A. Hirst, and H. Fujii, Regulation of cell proliferation by interleukin-3-induced nuclear translocation of pyruvate kinase. J Biol Chem, 2007. 282(24): p. 17706-11.
40. Ignacak, J. and M.B. Stachurska, The dual activity of pyruvate kinase type M2 from chromatin extracts of neoplastic cells. Comp Biochem Physiol B Biochem Mol Biol, 2003. 134(3): p. 425-33.
41. Lee, J., et al., Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol, 2008. 40(5): p. 1043-54.
42. Gao, X., et al., Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell, 2012. 45(5): p. 598-609.
43. Wang, H.J., et al., JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism. Proc Natl Acad Sci U S A, 2014. 111(1): p. 279-84.
44. Yang, W., et al., PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell, 2012. 150(4): p. 685-96.
45. Anastasiou, D., et al., Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol, 2012. 8(10): p. 839-47.
46. Arora, A. and S. Maiti, Differential biophysical behavior of human telomeric RNA and DNA quadruplex. J Phys Chem B, 2009. 113(30): p. 10515-20.
47. Gupta, V., et al., Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy. J Biol Chem, 2010. 285(22): p. 16864-73.
48. Iqbal, M.A., et al., Missense mutations in pyruvate kinase M2 promote cancer metabolism, oxidative endurance, anchorage independence, and tumor growth in a dominant negative manner. J Biol Chem, 2014. 289(12): p. 8098-105.
49. Otwinowski, Z. and W. Minor, Processing of X-ray diffraction data collected in oscillation mode. Macromol Crystallography, Pt A, 1997. 276: p. 307-326.
50. Murshudov, G.N., A.A. Vagin, and E.J. Dodson, Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D-Biological Crystallography, 1997. 53: p. 240-255.
51. Lebedev, A.A., A.A. Vagin, and G.N. Murshudov, Model preparation in MOLREP and examples of model improvement using X-ray data. Acta Crystallogr, Sect D: Biol Crystallogr, 2008. 64: p. 33-39.
52. Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr, Sect D: Biol Crystallogr, 2004. 60: p. 2126-2132.
53. Bailey, S., The Ccp4 Suite - Programs for Protein Crystallography. Acta Crystallogr, Sect D: Biol Crystallogr, 1994. 50: p. 760-763.
54. Murshudov, G.N., et al., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr, Sect D: Biol Crystallogr, 2011. 67: p. 355-367.
55. Evrard, G.X., et al., Assessment of automatic ligand building in ARP/wARP. Acta Crystallogr, Sect D: Biol Crystallogr, 2007. 63: p. 108-117.
56. Laskowski, R.A., et al., Procheck - a Program to Check the Stereochemical Quality of Protein Structures. IJACT, 1993. 26: p. 283-291.
57. Larkin, M.A., et al., Clustal W and clustal X version 2.0. Bioinformatics, 2007. 23(21): p. 2947-2948.
58. Krissinel, E. and K. Henrick, Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 12 Pt 1): p. 2256-68.
59. DeLano, W.L., Use of PYMOL as a communications tool for molecular science. Abstracts of Papers of the American Chemical Society, 2004. 228: p. U313-U314.
60. Murshudov, G.N., et al., Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallographica Section D-Biological Crystallography, 1999. 55: p. 247-255.
61. Morgan, H.P., et al., M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation. Proc Natl Acad Sci U S A, 2013. 110(15): p. 5881-6.