BackgroundThe gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein.ResultsThe addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure.ConclusionsPurification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.

中文翻译：

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表位标签改变磷酸甘油酸脱氢酶结构并削弱支持细胞增殖的能力

背景编码丝氨酸生物合成途径酶 PHGDH 的基因位于人类癌症中焦点基因组拷贝数增加的区域。具有 PHGDH 扩增的细胞依赖于酶表达进行增殖。然而，对增加的 PHGDH 表达的依赖超出了单独生产丝氨酸的范围，需要进一步研究 PHGDH 功能以阐明其在癌细胞中的作用。这些研究将需要一种生理相关形式的酶，用于使用工程细胞系和重组蛋白的实验。结果向 PHGDH 添加 N 端表位标签消除了支持 PHGDH 扩增细胞增殖的能力，尽管保留了一些转化活性3-PG 转 PHP。将 R236E 突变引入 PHGDH 会消除酶活性，并且这种催化失活的酶不能支持 PHGDH 依赖性细胞的增殖，认为需要典型的酶活性。标记和未标记的 PHGDH 表现出相同的细胞内定位和产生 D-2-羟基戊二酸 (D-2HG) 的能力，这是 PHGDH 的错误产物，认为错误定位或 D-2HG 产生的损失都不能解释表位标记的 PHGDH 不能支持扩散。为了能够研究 PHGDH 功能，我们报告了一种纯化重组 PHGDH 的方法，发现未标记的酶活性大于 N 末端标记的酶。使用尺寸排阻色谱法和电子显微镜分析标记和未标记的 PHGDH 发现 N 端表位标记改变酶结构。结论未标记的重组 PHGDH 的纯化消除了使用表位标签进行酶研究的需要。此外，虽然标记的 PHGDH 保留了一些将 3PG 转化为 PHP 的能力，但由包含表位标记引起的结构改变破坏了 PHGDH 维持癌细胞增殖的能力。