Background Many cancers silence the metabolic enzyme argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme for arginine biosynthesis within the urea cycle. Consequently, ASS1-negative cells are susceptible to depletion of extracellular arginine by PEGylated arginine deiminase (ADI-PEG20), an agent currently being developed in clinical trials. As the primary mechanism of resistance to arginine depletion is re-expression of ASS1, we sought a tool to understand the temporal emergence of the resistance phenotype at the single-cell level. Methods A real-time, single-cell florescence biosensor was developed to monitor arginine-dependent protein translation. The versatile, protein-based sensor provides temporal information about the metabolic adaptation of cells, as it is able to quantify and track individual cells over time. Results Every ASS1-deficient cell analyzed was found to respond to arginine deprivation by decreased expression of the sensor, indicating an absence of resistance in the naïve cell population. However, the temporal recovery and emergence of resistance varied widely amongst cells, suggesting a heterogeneous metabolic response. The sensor also enabled determination of a minimal arginine concentration required for its optimal translation. Conclusions The translation-dependent sensor developed here is able to accurately track the development of resistance in ASS1-deficient cells treated with ADI-PEG20. Its ability to track single cells over time allowed the determination that resistance is not present in the naïve population, as well as elucidating the heterogeneity of the timing and extent of resistance. This tool represents a useful advance in the study of arginine deprivation, while its design has potential to be adapted to other amino acids.

中文翻译：

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细胞内精氨酸依赖性翻译传感器揭示了 ASS1 阴性细胞中精氨酸饥饿反应和抵抗的动态

背景 许多癌症使代谢酶精氨琥珀酸合成酶 1 (ASS1) 沉默，这是尿素循环中精氨酸生物合成的限速酶。因此，ASS1 阴性细胞容易被聚乙二醇化精氨酸脱亚胺酶 (ADI-PEG20) 消耗掉细胞外精氨酸，该酶目前正在临床试验中开发。由于抗精氨酸消耗的主要机制是 ASS1 的重新表达，我们寻求一种工具来了解单细胞水平上抗性表型的时间出现。方法开发了一种实时单细胞荧光生物传感器来监测精氨酸依赖性蛋白质翻译。多功能、基于蛋白质的传感器提供有关细胞代谢适应的时间信息，因为它能够随着时间的推移量化和跟踪单个细胞。结果发现分析的每个 ASS1 缺陷细胞都通过传感器表达降低对精氨酸剥夺做出反应，表明幼稚细胞群中不存在抗性。然而，时间恢复和耐药性的出现在细胞之间差异很大，表明存在异质的代谢反应。该传感器还能够确定其最佳翻译所需的最低精氨酸浓度。结论 这里开发的依赖翻译的传感器能​​够准确跟踪用 ADI-PEG20 处理的 ASS1 缺陷细胞中耐药性的发展。随着时间的推移跟踪单个细胞的能力允许确定初始群体中不存在抗性，并阐明抗性时间和程度的异质性。