Characterization of Single Nucleotide Polymorphisms (SNPs) and Single Nucleotide Variations (SNVs) Mutants of Human Mitochondrial NAD(P)+-Dependent Malic Enzyme
| Autor: | Ting-Jhen Huang, 黃亭臻 |
|---|---|
| Rok vydání: | 2019 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 107 In mammals, malic enzyme (ME) can be divided into three isoforms according to their coenzyme specificity and subcellular localization: cytosolic NADP+-dependent isoform (c-NADP-ME, ME1), mitochondrial NAD+-dependent isoform (m-NAD-ME, ME2), and mitochondrial NADP+-dependent isoform (m-NADP-ME, ME3). Human ME2 is a mitochondrial NAD+-dependent malic enzyme, which is a homotetrameric protein and catalyzes an oxidative decarboxylation of malate to pyruvate. Comparing to ME1 and ME3, ME2 has an allosteric site for fumarate to regulate the enzyme activity, and uses both NAD+ and NADP+ as the coenzyme. In structure, ME2 has four configurations: open form I, II and closed form I, II, is according to the combination of ligands and fumarate for configuration convertion. In recent years, ME2 has been considered as an important enzyme in tumer energy metabolism, which is involved in the growth of cancer cells. ME2 widely presents in the cellular mitochondria granules, and we suspected that many mutations of ME2 have effects on cancer cells. Therefore, the enzyme kinetics of 30 SNPs were detected according the SNP database of dbSNP. However, with the update of the database, next-generation sequencing (NGS) in 2018 made the whole genome and transcriptome single nucleotide position in cancer were found. Point mutation (SNV) contained some SNPs and other lower frequency mutation sites in SNV. After arranging the database, 25 SNVs in each cancer were tested and compared with the ME2 wild type, including A39S, S77T, and E93D in the allosteric site; I104M, M108V, R165I, G168D, A293V, G446C, P472S, and G510W in the active site; V143F, A157V, R245I, F541L in exo-site; the stabilization of dimer or tetramer structure is L48I, S136L, Y570F, and distributed around the non-specific region Q335K, H367Y, S372R, A408T, T423A, and E438Q. After the analysis of enzyme kinetics, the ME2 inhibitor-embonic acid was used to identify the inhibition ability. Finally, the analytical ultracentrifuge (AUC) was used to observe the quaternary structure change. The changes of protein structure and enzyme activity could explore the relationship between these SNVs in cancers. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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