Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5′-deoxy-5′-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

甲基硫代腺苷磷酸化酶 (MTAP) 是蛋氨酸回收途径中的关键酶，可将多胺合成副产物 5'-deoxy-5'-methylthioadenosine (MTA) 转化为蛋氨酸。MTAP 的失活，通常是纯合缺失，在实体和血液系统恶性肿瘤中都有发现，并且是人类癌症中最常观察到的基因改变之一。以前的工作确定 MTAP 缺失的细胞会积累 MTA 并含有减少量的具有对称二甲基精氨酸 (sDMA) 的蛋白质。这些发现导致了这样的假设，即细胞内 MTA 的积累抑制了负责大量蛋白质 sDMA 酰化的蛋白质精氨酸甲基化酶 (PRMT5)。在这里，我们确认 MTAP 缺失的细胞增加了 MTA 积累并减少了蛋白质 sDMAylation。然而，+细胞，即使由于 MTAP 对 MTA 的分解代谢，未发现细胞内 MTA 持续增加。我们确定 MTA 对蛋白质 sDMAylation 的抑制发生在 48 小时内，是可逆的，并且是特异性的。此外，我们已经确定了两种增强子结合蛋白 FUBP1 和 FUBP3，它们在响应 MTAP 和 MTA 时差异 sDMAylated。这些蛋白质通过位于 Myc 和其他启动子上游的远上游元件位点。使用包含远上游元素位点的转录报告构建体，我们证明添加 MTA 可以减少转录，这表明 FUBP1 和 FUBP3 sDMAylation 的减少具有功能性后果。总体而言，我们的研究结果表明，细胞外 MTA 可以抑制蛋白质 sDMAylation，并且这种抑制会影响 FUBP 功能。