Homeostatic Regulation of Folate Metabolism

描述(申请人提供)：这项研究旨在阐明细胞核叶酸代谢的调节和功能，并确定其在细胞培养和基因敲除动物模型中对基因组完整性的贡献。维生素缺乏和/或单核苷酸多态破坏了叶酸代谢，影响了ADOMet和DTMP的合成，从而影响了DMA中染色质甲基化和尿嘧啶的含量。尿嘧啶和甲基化都影响DNA的稳定性，甲基化调节许多基因的表达。目前尚不清楚叶酸代谢与病理(包括某些癌症、心血管疾病)和发育异常(包括神经管缺陷)之间的关联是否源于改变的ADOMet和/或DTMP合成。最近，我们发现胞浆丝氨酸羟甲基转移酶(CSHMT)是一种代谢开关，它引导叶酸激活的一碳单位在DTMP和ADOMet生物合成途径之间流动。CSHMT在与叶酸相关的病理组织中表达，包括发育中的神经管和结肠；它的表达受几种营养物质的动态调节。因此，cSHMT可能在叶酸相关病理的病因中起作用，并成为饮食预防的靶点。最近，我们发现在细胞周期的S期，cSHMT定位于细胞核，并被相扑修饰。这些实验的目的是在cSHMT表达和总甲基化与核叶酸代谢之间，以及核叶酸代谢与基因组完整性之间进行直接和一致的翻译。阐明叶酸核代谢的功能将增加我们对叶酸相关病理的认识。其具体目的是：1.确定叶酸核代谢的存在及其影响其效率的因素。II.确定人cSHMT L474F SNP对核叶酸代谢的影响。III.确定cSHMT表达降低影响TS蛋白水平的机制。确定叶酸缺乏诱导肠道cSHMT蛋白水平的机制。需要检验的主要假设是：1.cSHMT通过与TS和DHFR的核共定位促进DTMP的合成。2.和甲基化是叶酸核代谢所必需的。3.核叶酸代谢增强基因组稳定性。4.TS和cSHMT的表达是协同调控的。5.叶酸/胆碱对cSHMT的调节保留了DTMP池，但损害了ADOMet的合成。该项目的长期目标是：1.确定利用BV细胞调节叶酸代谢的动态平衡机制。

DESCRIPTION (provided by applicant): This research aims to elucidate the regulation and function of nuclear folate metabolism and determine its contribution to genome integrity in both cell culture and knock-out animal models. Disruption of folate metabolism by vitamin deficiency and/or single nucleotide polymorphisms affects AdoMet and dTMP syntheses and thereby influences chromatin methylation and uracil content in DMA. Both uracil and methylation affect DNA stability, and methylation regulates the expression of many genes. It is not known if the associations between folate metabolism and pathologies (including certain cancers, cardiovascular disease) and developmental anomalies (including neural tube defects) result from altered AdoMet and/or dTMP syntheses. Recently, we showed that the enzyme cytoplasmic serine hydroxymethyltransferase (cSHMT) is a metabolic switch that directs the flux of folate-activated one-carbon units between the dTMP and AdoMet biosynthetic pathways. cSHMT is expressed in tissues associated with folate-related pathologies including the developing neural tube and colon; its expression is dynamically regulated by several nutrients. Therefore, cSHMT may contribute to the etiology of folate-related pathologies and a target for prevention through diet. Recently, we discovered that cSHMT localizes to the nucleus during the S phase of the cell cycle and that it modified by SUMO. These experiments are designed to make direct and coherent translations between cSHMT expression & sumoylation and nuclear folate metabolism, and between nuclear folate metabolism and genome integrity. Elucidating the function of nuclear folate metabolism will increase our understanding of folate-related pathologies. The specific aims are: I. to determine the existence of nuclear folate metabolism and the factors that influence its efficiency. II. to determine the impact of the human cSHMT L474F SNP on nuclear folate metabolism. III. to determine the mechanisms whereby decreases in cSHMT expression affect TS protein levels. IV. to determine the mechanism whereby folate deficiency induces cSHMT protein levels in the intestine. The principle hypotheses to be tested are that: 1. cSHMT enhances dTMP synthesis through nuclear co-localization with TS & DHFR. 2. sumoylation is essential for nuclear folate metabolism. 3. nuclear folate metabolism enhances genome stability. 4. TS and cSHMT expression are coordinately regulated. 5. regulation of cSHMT by folate/choline preserves dTMP pools at the expense of AdoMet synthesis. The long-term goals of this project are: 1. to define homeostatic mechanisms used bv cells to regulate folate metabolism.