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）是蛋氨酸补救途径中的关键酶，其功能是 将5 ′-脱氧-5 ′-甲硫基腺苷（MTA）转化为甲硫氨酸。MTAP的灭活，通常通过 纯合缺失，发现于实体和血液恶性肿瘤，是最常见的 在人类癌症中观察到基因改变。我们实验室和其他人以前的工作已经确定MTAP 可以作为肿瘤抑制基因。然而，MTAP损失促进 肿瘤发生仍不清楚。一种可能的机制涉及MTA的积累，MTA是由 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的大规模遗传筛选已经确定MTAP- 缺失的细胞对特定蛋白质精氨酸甲基转移酶的敲低特别敏感 （PRMT5），其负责精氨酸的翻译后对称二甲基化修饰 残基（sDMA）。这种修饰经常在参与mRNA成熟的蛋白质中观察到。在 初步数据，我们证明，MTAP的损失或细胞外MTA的增加导致显着减少， 含sDMA蛋白质的稳态水平。值得注意的是，当加入细胞外MTA时， 细胞内MTA增加，表明sDMA-化的减少是由于信号转导 过程然而，我们的数据还表明，不依赖于MTA， 可影响mRNA水平并拮抗MTA的作用。因此，本提案的总体目标是探讨 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 另一种是作为癌代谢物，另一种是直接作用于MTAP蛋白。本研究的具体目的是：（1） 鉴定响应MTAP而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在癌细胞中sDMA裂解机制中的作用;以及 (3)阐明MTAP的酶功能与非酶功能。这些研究意义重大，因为 它们为理解MTAP缺失在肿瘤发生中的作用提供了潜在的机制， 可能会导致新的治疗策略MTAP缺失的肿瘤。此外，这些研究将揭示 "管家"代谢酶如何作为肿瘤抑制基因发挥新作用。

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.