A Novel Function for G6PD in Regulation of the Cancer Epigenome

描述(申请人提供)：葡萄糖-6-磷酸脱氢酶(G6PD)是磷酸戊糖途径的限速酶，是细胞产生还原等效物的主要代谢途径。在氧化还原反应中，G6PD氧化葡萄糖-6-磷酸，并将电子转移到烟酰胺腺嘌呤二核苷酸磷酸(NADP+)，生成NADPH。NADPH为生物合成途径提供还原能量，这些途径产生大分子，如肝脏或乳腺中核酸和脂肪酸的合成。NADPH在维持细胞的抗氧化能力方面也是必不可少的。G6PD通常被认为是一种细胞质酶，其活性受NADPH/NADP+比率的调节。据报道，从心肌病到癌症，许多疾病都有G6PD活性升高的报道。我们最近发现，NADPH，而不是NADP+，NADH或NAD+，在体外是I类组蛋白脱乙酰酶(HDACs)1和2的变构激活剂。NADPH通过混合激活动力学来激活HDAC，增加了HDAC酶对其组蛋白底物的亲和力以及脱乙酰化反应的速度。为了在体内支持我们的发现，我们推测，药物抑制细胞内的G6PD应该会导致NADPH水平下降，进而降低HDAC活性，增加组蛋白乙酰化。事实上，我们观察到，在MDA-MB-453乳腺癌细胞中抑制G6PD会使全局组蛋白乙酰化增加多达三倍。另一种乳腺癌细胞株，MDA-MB-231，对G6PD抑制没有表现出同样的反应。进一步的研究发现，与MDA-MB-231相比，G6PD在MDA-MB-453细胞中高表达，并且可检测到的G6PD部分定位于细胞核，进一步的部分与染色质显著结合。自从G6PD被发现以来的80年里，只有两个报告表明G6PD可能存在于细胞核中，而在染色质上没有。此外，我们的初步CHIP-SEQ分析表明，G6PD与染色质的结合不是虚假的，而是与与钙稳态相关的功能基因接近-这是乳腺癌生物学中的一条重要途径。在这个应用中，我们认为G6PD与染色质的结合揭示了G6PD的一个额外和重要的功能，这一功能尚未在任何程度上得到表征。我们进一步推测，G6PD可能在特定的基因组座位上发挥作用，在局部产生NADPH，从而激活HDAC和组蛋白去乙酰化。我们现在的目标是确定G6PD在整个基因组中的分布及其与HDAC结合、组蛋白乙酰化和基于染色质的过程(如基因表达)调节的功能相关性。我们相信，我们的数据将为研究这种关键代谢酶的功能开辟一个新的领域，并将揭示 基因组的新陈代谢和表观遗传调控的显著交汇点。

DESCRIPTION (provided by applicant): Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway, the major metabolic pathway for generation of reducing equivalents for the cell. In an oxidation- reduction reaction, G6PD oxidizes glucose-6-phosphate and transfers the electrons to nicotinamide adenine dinucleotide phosphate (NADP+) to generate NADPH. NADPH provides reducing power for biosynthetic pathways that generate macromolecules such as synthesis of nucleic and fatty acids in the liver or the mammary glands. NADPH is also essential in maintaining antioxidant capacity of the cell. G6PD is normally considered to be a cytoplasmic enzyme where its activity is regulated by NADPH/NADP+ ratio. Increased G6PD activity has been reported for many diseases ranging from cardiomyopathy to cancers. We have recently discovered that NADPH, but not NADP+, NADH or NAD+, is an allosteric activator of class I histone deacetylases (HDACs) 1 and 2 in vitro. NADPH activates HDACs through a mixed activation kinetic, increasing the affinity of the HDAC enzyme for its histone substrate as well as the velocity of the deacetylation reaction. To provide in vivo support for our findings, we surmised that pharmacological inhibition of G6PD inside cells should lead to decreased NADPH levels and in turn to decreased HDAC activity and increased histone acetylation. Indeed, we observed that G6PD inhibition in MDA-MB-453 mammary cancer cells increases global histone acetylation by up to three fold. Another breast cancer cell lines, MDA-MB-231, did not show the same response to G6PD inhibition. Further investigation revealed that G6PD is highly expressed in MDA-MB-453 cells compared to MDA-MB-231 and that a detectable fraction of G6PD is localized in the nucleus with a further fraction strikingly bound to chromatin. In the 80 years since G6PD was discovered, only two reports indicate a potential presence of G6PD in the nucleus and none on chromatin. Moreover, our preliminary ChIP-seq analysis has revealed that G6PD binding to chromatin is not spurious but rather near genes with functions related to calcium homeostasis-an important pathway in breast cancer biology. In this application, we propose that G6PD binding to chromatin reveals an additional and significant function of G6PD that has not been characterized to any extent. We further postulate that G6PD may function at specific genomic loci to produce NADPH locally for HDAC activation and histone deacetylation. We now aim to determine the distribution of G6PD across the genome and its functional relevance to HDAC binding, histone acetylation and regulation of chromatin based processes such as gene expression. We believe our data will open up a new field of inquiry into the function of this critical metabolic enzyme and will uncover a remarkable juncture where metabolism and epigenetic regulation of the genome intersect.