Posttranslational regulation of augurin, a new secretory tumor suppressor

描述（由申请人提供）：胶质母细胞瘤是成人中最常见的原发性恶性脑肿瘤类型，也是最具侵袭性的癌症之一。尽管这种类型癌症的手术治疗最近取得了进展，但这些患者的中位生存时间约为14个月，这很可能是由于肿瘤基因组中肿瘤抑制基因表达的丧失。监测血浆或脑活检中某些分子的水平可能为我们提供有关早期可能的胶质母细胞瘤发展/进展的信息。Augurin，最近被鉴定为潜在的肿瘤抑制剂，是垂体、脑、甲状腺中产生的分泌分子， 和食道。它参与了从ACTH释放到肿瘤抑制的广泛的生理过程，并且在所有脊椎动物中非常保守。最有趣的是，Augurin的表达在某些类型的癌症（包括胶质母细胞瘤）中通过启动子超甲基化而下调。分泌蛋白参与多种生理功能，如调节代谢、细胞增殖、分化和细胞死亡;它们在通过分泌途径运输期间需要许多翻译后修饰以获得生物活性状态。没有这些翻译后修饰，分泌蛋白就不能发挥其生物学活性，导致生理功能的缺失。然而，关于各种细胞类型中内源性形式的原Augrin衍生肽的信息尚未报道。这个提议的目的是了解proaugurin作为一种肿瘤抑制因子的翻译后修饰。我们最近的报告表明，两个特定的proaugurin衍生的肽产生的特定前蛋白转化酶弗林蛋白酶在体外的行动。此外，我们已经发现，proaugurin是硫酸化的贩运过程中通过分泌途径。胶质母细胞瘤细胞系的增殖试验表明，只有弗林蛋白酶裂解的proaugurin可以抑制细胞增殖，这表明蛋白水解裂解是proaugurin表现出生物活性的翻译后要求。此外，Augurin中预测的硫酸化位点高度 在其他物种中是保守的，支持Augurin中的硫酸化可能有助于抑制肿瘤细胞增殖的活性的观点。在这个提议中，我们将确定Augurin的生物活性种类，其功能是使用其合成的抑制肿瘤细胞增殖。 原Augurin衍生肽的肽;进行质谱分析以确定Augurin中的硫酸化位点并阐明Augurin中硫酸化的重要性;确定Augurin水平 在正常和神经胶质瘤荷瘤小鼠中使用放射免疫测定和免疫组织化学染色，以测试降低Augurin表达可能是肿瘤发展细胞的潜在指标的想法。这些实验将有助于揭示proaugurin成熟的分子机制，并将提供线索，其作为一种肿瘤抑制剂的生物学意义。

DESCRIPTION (provided by applicant): Glioblastoma is the most common type of primary malignant brain tumor in adults and one of the most aggressive cancers. Even though there have been recent advances in surgical management of this type of cancer, the median survival time for these patients is approximately 14 months, most likely due to the loss of tumor suppressor expression within the tumor genome. Monitoring the level of certain molecules in either plasma or brain biopsies might provide us with information regarding the development/progression of a possible glioblastoma in early stage. Augurin, recently identified as a potential tumor suppressor, is a secretory molecule produced in the pituitary, brain, thyroid, and esophagus. It is implicated in a wide array of physiological processes, from ACTH release to tumor suppression, and is well conserved among all vertebrates. Most interestingly, the expression of augurin is down-regulated by promoter hypermethylation in certain types of cancers, including glioblastoma. Secretory proteins are involved in diverse physiological functions, such as the regulation of metabolism, cell proliferation, differentiation, and cell deat; they require a number of posttranslational modifications during their trafficking through the secretory pathway in order to attain bioactive status. Without these posttranslational modifications, secretory proteins cannot exert their biological activities, resulting in deficiencis in physiological functions. However, information regarding the endogenous forms of proaugurin-derived peptides in various cell types has yet to be reported. The goal of this proposal is to understand the posttranslational modifications of proaugurin as a tumor suppressor. Our recent report has shown that two specific proaugurin-derived peptides are generated by the action of the specific proprotein convertase furin in vitro. Furthermore, we have found that proaugurin is sulfated during trafficking through the secretory pathway. Proliferation assays with a glioblastoma cell line demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to exhibit bioactivity. In addition, the predicted sulfation sites in augurin are highly conserved among other species, supporting the idea that sulfation in augurin might contribute to the suppressive activity against tumor cell proliferation. In this proposal, we will determine the bioactive species of augurin that function to suppress tumor cell proliferation using its synthetic peptides of proaugurin-derived peptides; perform mass spectrometry to determine the sulfation site(s) in augurin and clarify the importance of sulfation in augurin; determine the augurin levels in normal and glial tumor-bearing mice using radioimmunoassay and immunohistochemical staining, to test the idea that lowered augurin expression may be a potential indicator of tumor-developing cells. These experiments will help to reveal molecular mechanisms involved in proaugurin maturation, and will provide clues to its biological importance as a tumor suppressor.