MTAP, 5'-deoxy-5'-methylthioadenosine, and the dysregulation of symmetric dimethylarginine in cancer

项目摘要 甲硫腺苷磷酸化酶(MTAP)是蛋氨酸挽救途径中的一个关键酶，其功能 是将5‘-脱氧-5’-甲硫腺苷(MTA)转化为蛋氨酸。停用MTAP，通常是通过 纯合子缺失，在实体和血液系统的恶性肿瘤中都可发现，是最常见的 观察到人类癌症的基因变化。我们实验室和其他实验室之前的工作已经确定，MTAP 可以作为肿瘤抑制基因。然而，MTAP损失促进的确切机制 肿瘤发生机制仍不清楚。一种可能的机制涉及MTA的积累，MTA由 MTAP-缺失的肿瘤细胞。在结构上，MTA与腺苷非常相似，有证据表明它可以 与腺苷受体相互作用。使用shRNA的大规模遗传筛查已经确定MTAP- 被删除的细胞对一种特定蛋白质精氨酸甲基转移酶的敲除特别敏感 (PRMT5)，负责精氨酸的翻译后对称二甲基化修饰 残留物(SDMA)。这种修饰经常出现在与mRNA成熟有关的蛋白质中。在……里面 初步数据表明，MTAP的丢失或细胞外MTA的加入会导致显著的减少 含有SDMA的蛋白质的稳态水平。值得注意的是，当加入胞外MTA时，没有 细胞内MTA的增加，表明SDMA甲基化的减少是由于信号转导 进程。然而，我们的数据也表明，酶失活的MTAP蛋白本身，独立于MTA， 可影响mR-NA水平，拮抗MTA的作用。因此，这项提案的总体目标是探索 假设MTAP通过两种不同的机制介导其肿瘤抑制功能：一种涉及MTA 作为一种代谢物，另一种直接作用于MTAP蛋白。研究的具体目的是：(1) 确定对MTAP有差异甲基化的特定蛋白质和精氨酸残基；(2) 确定细胞内和细胞外MTA在癌细胞SDMA裂解机制中的作用；以及 (3)明确MTAP的酶与非酶功能。这些研究具有重要意义，因为 它们为理解MTAP缺失在肿瘤发生和发展中的作用提供了一种潜在的机制。 可能导致MTAP缺失肿瘤的新治疗策略。此外，这些研究将有助于揭示 “看家”代谢酶如何作为肿瘤抑制基因发挥新的作用。

Project Summary Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway whose function is to convert 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 from our lab and others has established that MTAP can act as a tumor suppressor gene. However the precise mechanism by which MTAP loss promotes tumorigenesis is still unclear. One possible mechanism involves the accumulation of MTA, which is secreted by MTAP-deleted tumor cells. Structurally, MTA closely resembles adenosine and evidence indicates that it can interact with adenosine receptors. Large-scale genetic screens using shRNA have established that MTAP- deleted cells are especially sensitive to knockdown of a specific protein arginine methyltransferase enzyme (PRMT5), which is responsible for the post-translational symmetric dimethylation modification of arginine residues (sDMA). This modification is frequently observed in proteins involved in mRNA maturation. In preliminary data, we demonstrate that loss of MTAP or addition of extracellular MTA causes a dramatic reduction of the steady-state levels of sDMA-containing proteins. Significantly, when extracellular MTA is added, no increase in intracellular MTA occurs, suggesting that the reduction in sDMA-ylation is due to a signal transduction process. However, our data also suggests that enzymatically inactive MTAP protein itself, independent of MTA, can affect mRNA levels and antagonize the effects of MTA. Thus the overall goal of this proposal is to explore the hypothesis MTAP mediates its tumor suppressor function via two different mechanisms: one involving MTA as an oncometabolite and the other a direct role for the MTAP protein. The specific aims of the study are: (1) Identify specific proteins and arginine residues that are differentially methylated in response to MTAP; (2) Determine the roles of intracellular and extracellular MTA in the mechanism of sDMA-lyation in cancer cells; and (3) Clarification of the enzymatic vs. non-enzymatic functions of MTAP. These studies are significant because they provide a potential mechanism for understanding the role that MTAP deletion plays in tumorigenesis and may lead to novel therapeutic strategies for MTAP-deleted tumors. In addition, these studies will shed light on how a “housekeeping” metabolic enzyme can have a novel role as a tumor suppressor gene.