The loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT). We determined growth rates and survival of cell lines in response to amino acid depletion and supplementation. RNAseq was used to determine the transcription changes contingent on Asn and Gln supplementation, which was further followed with stable isotope resolved metabolomics (SIRM) using both [U- 13C,15N] Gln and Asn. We found that Asn increased the growth rate of both cell lines in vitro. Gln, but not Asn, increased oxygen consumption rates and glycolytic reserve of both cell lines. Although Asn was taken up by the cells, there was little evidence of Asn-derived label in cellular metabolites, indicating that Asn was not catabolized. However, Asn strongly stimulated Gln labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway (HBP) by Asn. Asn in combination with Gln, but not Asn or Gln alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. Asn exposure also increased both the RNA and protein expression of the HBP regulator GFPT2, which is a transcriptional target of sXBP1. Asn in the presence of Gln induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of HBP activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.

中文翻译：

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富马酸水合酶缺陷型肾细胞癌细胞通过激活未折叠蛋白反应和刺激己糖胺生物合成途径对天冬酰胺作出反应

富马酸水合酶 (FH) 的功能丧失突变是遗传性平滑肌瘤病和肾细胞癌 (HLRCC) 的驱动因素。富马酸盐的积累导致应激相关机制的激活，从而导致细胞存活相关基因的上调。为了更好地了解细胞如何补偿 HLRCC 中 FH 的损失，我们确定了 FH 缺陷型 UOK262 细胞系 (UOK262) 及其 FH 补充对照 (UOK262WT) 的氨基酸营养需求。我们确定了响应氨基酸消耗和补充的细胞系的生长速率和存活率。RNAseq 用于确定取决于 Asn 和 Gln 补充的转录变化，随后使用 [U-13C,15N] Gln 和 Asn 进行稳定同位素解析代谢组学 (SIRM)。我们发现 Asn 在体外增加了两种细胞系的生长速率。Gln，但不是 Asn，增加了两种细胞系的耗氧率和糖酵解储备。虽然 Asn 被细胞吸收，但在细胞代谢物中几乎没有 Asn 衍生标记的证据，表明 Asn 没有被分解代谢。然而，Asn 强烈刺激 UOK262 细胞中的尿嘧啶和前体、磷酸尿苷和己糖胺代谢物的 Gln 标记，并且在 UOK262WT 细胞中的程度要小得多，表明 Asn 激活了己糖胺生物合成途径 (HBP)。Asn 与 Gln 组合，但不是 Asn 或 Gln 单独，刺激与 UOK262 中内质网 (ER) 应激和未折叠蛋白反应 (UPR) 相关的基因表达比在 FH 恢复细胞中更大。这些基因的表达变化通过 RT-PCR 得到证实，UPR 的刺激通过在这些条件下 UOK262 细胞中剪接 XBP1 (sXBP1) 的增加得到正交证实。Asn 暴露也增加了 HBP 调节因子 GFPT2 的 RNA 和蛋白质表达，GFPT2 是 sXBP1 的转录靶标。在 Gln 存在的情况下，Asn 在 FH 缺陷型 UOK262 细胞中诱导 ER 应激反应，并刺激 UDP-乙酰聚糖的合成增加，表明 HBP 活性。这些数据证明了天冬酰胺对 FH 缺陷细胞中细胞代谢的新作用，可以在治疗上加以利用。Asn 暴露也增加了 HBP 调节因子 GFPT2 的 RNA 和蛋白质表达，GFPT2 是 sXBP1 的转录靶标。在 Gln 存在的情况下，Asn 在 FH 缺陷型 UOK262 细胞中诱导 ER 应激反应，并刺激 UDP-乙酰聚糖的合成增加，表明 HBP 活性。这些数据证明了天冬酰胺对 FH 缺陷细胞中细胞代谢的新作用，可以在治疗上加以利用。Asn 暴露也增加了 HBP 调节因子 GFPT2 的 RNA 和蛋白质表达，GFPT2 是 sXBP1 的转录靶标。在 Gln 存在的情况下，Asn 在 FH 缺陷型 UOK262 细胞中诱导 ER 应激反应，并刺激 UDP-乙酰聚糖的合成增加，表明 HBP 活性。这些数据证明了天冬酰胺对 FH 缺陷细胞中细胞代谢的新作用，可以在治疗上加以利用。