Osmotic Pressure and Crowding Effects on Folate Mediated One Carbon Metabolism

渗透压和拥挤对叶酸介导的一碳代谢的影响

• 批准号: 8689382
• 负责人: Elizabeth E Howell
• 金额: $ 32.25万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689382
• 关键词: Abbreviations; Address; Affect; Alcohols; Amines; Amino Acid Substitution; Amino Acids; Animals; Arthritis; Bacteria; Bacterial Drug Resistance; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological Assay; Bovine Serum Albumin; Calorimetry; Carbon; Cardiovascular Diseases; Cells; Characteristics; Coenzymes; Complex; Crowding; Cytoplasm; DHFR gene; Diet; Diffusion; Dihydrofolate Reductase; Dihydropteroate Synthase; Dimethyl Sulfoxide; Elements; Environmental Risk Factor; Enzymes; Epithelial; Equilibrium; Escherichia coli; Folate; Folic Acid Antagonists; Folylpolyglutamate synthase; Food; Genes; Genetic Models; Glutamates; Growth; Half-Life; Health; Human; Hydrophobicity; Hydrostatic Pressure; Hydroxymethyltransferases; In Vitro; Lead; Ligand Binding; Link; MTHFR gene; Malaria; Malignant Neoplasms; Measurement; Measures; Mediating; Metabolism; Methionine; Methotrexate; Methylenetetrahydrofolate reductase (NADPH); Modeling; Molecular Weight; Mutation; NADP; Nitric Oxide Synthase; Osmolalities; Osmolar Concentration; Osmotic Pressure; Oxidation-Reduction; Oxides; Pharmaceutical Preparations; Play; Polyethylene Glycols; Positioning Attribute; Pregnancy; Prokaryotic Cells; Proteins; Pterins; Research; Role; Scientist; Series; Serine; Solvents; Stress; System; Tail; Testing; Tetrahydrofolates; Text; Thymidylate Synthase; Time; Titrations; Trimethoprim; Trimethoprim Resistance; Vitamins; Walking; Water; adverse outcome; cofactor; dihydrofolate; fitness; folic acid metabolism; fortification; in vivo; macromolecule; mutant; nucleic acid metabolism; nutrition; protein function; protein structure function; public health relevance; research study; resistance factors; restoration; scaffold; small molecule; solute; trimethyloxamine

DESCRIPTION (provided by applicant): Previous studies in the Howell lab have found that addition of neutral osmolytes to an R-plasmid encoded dihydrofolate reductase (R67 DHFR) as well as the non-homologous E. coli chromosomal DHFR (EcDHFR) result in weaker binding of the substrate, dihydrofolate (DHF). This result contrasts with the tighter binding of NADPH under these same conditions. The cofactor results are more typical, as the decreased water content reduces the desolvation penalty associated with binding. A model to explain the weaker binding of DHF to DHFR hypothesizes that weak interactions occur between dihydrofolate and various osmolytes. If the DHF"osmolyte interaction is stronger than the DHF"water interaction, then it will take more energy to lose the DHF"osmolyte pair and binding of DHF to DHFR will be more difficult. (In other words, water prefers to interact with osmolytes as compared to folate.) To expand this solvent substitution hypothesis of enzyme action, different folate derivatives as well as different folate utilizing enzymes will be studied. Aim 1 proposes a series of experiments to study how DHF and folate derivatives interact with osmolytes. A Hansch plot will be used to determine if hydrophobicity is the signature element that makes folate "sticky." Aim 2 extends these biochemical studies to include other enzymes in one carbon metabolism. Aim 3 returns to DHFR to study the effects of macromolecular crowding on DHFR activity as the motifs found in osmolytes can also be found in proteins, leading to the proposal that DHF"crowder interactions will occur and affect catalytic efficiency. Aim 4 proposes a series of in vivo tests of the folate"osmolyte interaction model by genetic complementation assays performed under low water activity conditions in E. coli. Osmotic stress is predicted to result in lower catalytic efficiencies for enzymes involved in folate mediated one carbon metabolism, ultimately leading to blockage of growth. Aim 4B considers osmolyte effects on antibacterial resistance conferred by DHFR upon host E. coli and addresses what happens to the DHF and DHFR concentrations. This groundbreaking research identifies osmolality (or low water activity) as a key environmental factor involved in modulating DHFR function. By extension, other important folate utilizing enzymes will also be impacted. Factors that affect the folate, or vitamin B9, concentration, half-life and accessibility are important to human health as the redox forms of folate are substrates and coenzymes in the one carbon cycle, which impacts amino acid and nucleic acid metabolism. As animals do not synthesize folate or vitamin B9, they must obtain it from their diet. The consequences of insufficient folate nutrition can be seen in the adverse outcomes of pregnancy, thus the recent folate fortification of foods. Antifolates are also used as treatments o bacterial infections, malaria, cancer, arthritis, cardiovascular disease, etc. These are a few examples where folate biochemistry plays a role in human health.

描述(申请人提供)：Howell实验室以前的研究发现，向编码二氢叶酸还原酶(R67 DHFR)的R-质粒(R67 DHFR)以及非同源的大肠杆菌染色体DHFR(EcDHFR)添加中性渗透分子会导致底物二氢叶酸(DHF)的结合较弱。这一结果与相同条件下NADPH的更紧密结合形成了鲜明对比。辅因子的结果是更典型的，因为减少的水分含量减少了与结合相关的脱溶惩罚。一个解释DHF与DHFR结合较弱的模型假设二氢叶酸与各种渗透分子之间存在弱相互作用。如果DHF的渗透分子相互作用强于DHF的水相互作用，那么失去DHF渗透分子对需要更多的能量，DHF与DHFR的结合将更加困难。(换句话说，与叶酸相比，水更喜欢与渗透分子相互作用。)为了扩展这一酶作用的溶剂替代假说，我们将研究不同的叶酸衍生物以及不同的叶酸利用酶。目的1提出一系列实验来研究DHF和叶酸衍生物与渗透压的相互作用。Hansch图将被用来确定疏水性是否是使叶酸“粘性”的标志性元素。AIM 2扩展了这些生化研究，将一种碳代谢中的其他酶也包括在内。目的3回到DHFR来研究大分子拥挤对DHFR活性的影响，因为渗透分子中发现的基序也可以在蛋白质中找到，导致DHF的Crowder相互作用将发生并影响催化效率。目的4通过低水分活度条件下的遗传互补分析，对叶酸渗透分子相互作用模型进行了一系列体内试验。据预测，渗透胁迫会导致参与叶酸介导的一种碳代谢的酶的催化效率降低，最终导致生长受阻。目的4B研究渗透分子对DHFR对宿主大肠杆菌的抗菌活性的影响，并说明DHF和DHFR浓度的变化。这项开创性的研究确定渗透压(或低水分活度)是参与调节DHFR功能的关键环境因素。推而广之，其他重要的叶酸利用酶也将受到影响。影响叶酸或维生素B9浓度、半衰期和可及性的因素对人类健康很重要，因为叶酸的氧化还原形式是一个碳循环中的底物和辅酶，影响氨基酸和核酸的新陈代谢。由于动物不会合成叶酸或维生素B9，它们必须从饮食中获得叶酸。叶酸营养不足的后果可以从怀孕的不良后果中看出，因此最近在食物中添加叶酸。抗叶酸还被用于治疗细菌感染、疟疾、癌症、关节炎、心血管疾病等。这些都是叶酸生物化学在人类健康中发挥作用的几个例子。

项目成果

Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions.

Crowders 通过五元相互作用窃取二氢叶酸还原酶配体。

• DOI: 10.1021/acs.biochem.8b01110
• 发表时间: 2019
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: DuffJr,MichaelR;Desai,Nidhi;Craig,MichaelA;Agarwal,PratulK;Howell,ElizabethE
• 通讯作者: Howell,ElizabethE

In Vivo Titration of Folate Pathway Enzymes.

叶酸途径酶的体内滴定。

• DOI: 10.1128/aem.01139-18
• 发表时间: 2018
• 期刊: Applied and environmental microbiology
• 影响因子: 4.4
• 作者: Nambiar,Deepika;Berhane,Timkhite-Kulu;Shew,Robert;Schwarz,Bryan;DuffJr,MichaelR;Howell,ElizabethE
• 通讯作者: Howell,ElizabethE

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

• DOI: 10.1021/acs.biochem.5b00981
• 发表时间: 2016-01-12
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Duff, Michael R., Jr.;Chopra, Shaileja;Strader, Michael Brad;Agarwal, Pratul K.;Howell, Elizabeth E.
• 通讯作者: Howell, Elizabeth E.

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase.

• DOI: 10.1021/acs.jmedchem.0c00546
• 发表时间: 2020-08-13
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Duff MR Jr;Gabel SA;Pedersen LC;DeRose EF;Krahn JM;Howell EE;London RE
• 通讯作者: London RE

Thermodynamics and solvent linkage of macromolecule-ligand interactions.

• DOI: 10.1016/j.ymeth.2014.11.009
• 发表时间: 2015-04
• 期刊: METHODS
• 影响因子: 4.8
• 作者: Duff, Michael R., Jr.;Howell, Elizabeth E.
• 通讯作者: Howell, Elizabeth E.