Posttranslational regulation of augurin, a new secretory tumor suppressor

描述(申请人提供)：胶质母细胞瘤是成人最常见的原发恶性脑肿瘤类型，也是最具侵袭性的癌症之一。尽管最近在外科治疗这类癌症方面取得了进展，但这些患者的中位生存期约为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缺失。监测血浆或脑活检组织中某些分子的水平可能为我们提供有关胶质母细胞瘤早期发展/进展的信息。Augurin，最近被发现是一种潜在的肿瘤抑制因子，是一种在脑下垂体、脑、甲状腺产生的分泌分子， 还有食道。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都很保守。最有趣的是，在某些类型的癌症中，包括胶质母细胞瘤在内，奥古林的表达受到启动子超甲基化的下调。分泌蛋白参与多种生理功能，如新陈代谢、细胞增殖、分化和细胞死亡的调节；它们在通过分泌途径运输过程中需要进行一些翻译后修饰才能达到生物活性状态。如果没有这些翻译后修饰，分泌蛋白就不能发挥其生物活性，导致生理功能的缺陷。然而，关于不同细胞类型中的内源性前体多肽形式的信息还没有报道。这项建议的目的是了解前体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，在体外，通过特定的原蛋白转换酶Furin的作用，可以产生两个特定的原蛋白衍生的多肽。此外，我们还发现前体蛋白在通过分泌途径运输的过程中被硫酸盐化。对胶质母细胞瘤细胞系的增殖分析表明，只有呋喃裂解的前体蛋白才能抑制细胞的增殖，这表明蛋白水解性切割是前体蛋白显示生物活性的翻译后要求。此外，奥古林中预测的硫酸盐化部位高度 在其他物种中保守，支持奥古林中的硫酸盐化可能有助于抑制肿瘤细胞增殖的观点。在这项建议中，我们将确定奥古林的生物活性物种，其功能是通过其合成的抑制肿瘤细胞的增殖 前体多肽；进行质谱分析以确定奥古林中的硫化部位(S)，并阐明硫化在奥古林中的重要性；测定奥古林的水平 在正常和神经胶质瘤荷瘤小鼠中，使用放射免疫分析和免疫组织化学染色，测试Auurin表达降低可能是肿瘤细胞发展的潜在指标的想法。这些实验将有助于揭示与前体成熟有关的分子机制，并将为其作为肿瘤抑制因子的生物学重要性提供线索。

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.