Linking a deubiquitinating enzyme to the regulation of a human AlkB demethylation complex in cancer progression

通过脱烷基化保持DNA完整性是维持基因组稳定性的关键步骤。由于大多数烷基化损伤是内源性发生的，因此烷基化修复与维持基因组完整性非常相关，特别是对于高增殖组织如癌症。烷基化损伤主要由脱甲基酶/脱烷基酶直接修复。我们以前的研究结果表明，Abh 3，一种人类脱甲基酶/脱烷基酶复合物，通过烷基化修复促进癌症进展。前列腺肿瘤细胞中TAP纯化的Abh 3复合物纯化揭示了去泛素化酶的存在，称为卵巢肿瘤结构域OTUD 4。引人注目的是，随着时间的推移，用蛋白酶体抑制剂MG 132处理揭示了Abh 3的增加，这在对照中没有观察到。全细胞提取物的蛋白质印迹分析区分了癌细胞中Abh 3和OTUD 4的共表达，并且shRNA介导的OTUD 4敲低与体内（至少在癌细胞中）Abh 3表达降低相关。Abh 3和OTUD 4的敲低导致增殖降低以及烷基化损伤敏感性增加。因此，我们的研究结果表明，Abh 3的调节可能是通过泛素途径进行的，这可能是癌症特异性的，并且Abh 3和OTUD 4的相互作用可能促进肿瘤进展。这项拨款提案围绕着我们的主要假设，即OTUD 4通过将其去泛素化酶活性与人Abh 3去甲基化酶复合物联系起来来促进肿瘤进展。我们首先计划使用肿瘤组织阵列研究Abh 3和OTUD 4的共定位，并进一步表征Abh 3和OTUD 4的生化相互作用。其次，我们将阐明OTUD 4在体内DNA烷基化修复和肿瘤生长中的功能。第三，我们将研究OTUD 4是否在体外和体内使Abh 3去泛素化，并表征OTUD 4去泛素化酶活性的性质。第四，我们计划分析泛素结合对Abh 3的影响和特定的泛素化位点，并表征泛素连接的性质。第五，我们将证明OTUD 4去泛素化酶活性通过调节蛋白酶体介导的降解来调节Abh 3蛋白半衰期的细胞稳定性，并分析OTUD 4对Abh 3催化活性和对Abh 3介导的烷基化损伤修复的影响。第六，我们将研究我们的两种酶在各种烷化剂存在下对我们的癌症模型中的化疗耐药性的可能影响。总之，了解Abh 3和OTUD 4的协同作用可能有助于我们了解脱甲基酶/脱烷基酶的细胞表达是如何调节和维持的。脱烷基化酶的表达可能是癌细胞如何机械地采用烷基化损伤抗性的一种可能的解释，例如在用烷化剂化疗期间。拟议的项目可能使我们能够将DNA烷基化修复与表观遗传事件联系起来，并可能促进两个癌基因候选者Abh 3和OTUD 4成为未来潜在的抗癌靶点。

Maintaining DNA integrity by dealkylation is a crucial step in the maintenance of genomic stability. Since the majority of alkylation damage occurs endogenously, alkylation repair is of great relevance to maintain genomic integrity, in particular for high proliferating tissues like in cancer. Alkylation damage is mainly repaired directly by demethylases/dealkylases. Our previous findings suggest that Abh3, a human demethylase/dealkylase complex, promotes cancer progression via alkylation repair. TAP-purified Abh3 complex purification in prostate tumor cells reveals presence of a deubiquitinase, named Ovarian Tumor Domain OTUD4. Strikingly, treatment over time with the proteasome-inhibitor MG132 revealed an increase of Abh3, which is not seen in the controls. Western blot analysis of whole cell extracts demarks co-expression of Abh3 and OTUD4 in cancer cells and shRNAi-mediated knock down of OTUD4 is associated with decreased Abh3 expression in vivo, at least in cancer cells. Knock down of Abh3 and OTUD4 results in decreased proliferation as well as increased alkylation damage sensitivity. Therefore, our findings implicate that regulation of Abh3 may be performed via the ubiquitin pathway, which may be cancer specific, and interplay of Abh3 and OTUD4 may promote tumor progression. This grant proposal revolves around our primary hypothesis that OTUD4 promotes tumor progression by linking its deubiquitinase activity to the human Abh3 demethylase complex. We first plan to invest co-localization of Abh3 and OTUD4 using tumor tissue arrays and characterize further biochemical interaction of Abh3 and OTUD4. Second, we will elucidate the function of OTUD4 in DNA alkylation repair and tumor growth in vivo. Third, we will investigate if OTUD4 deubiquitinates Abh3 in vitro and in vivo and characterize the nature of OTUD4 deubiquitinase activity. Fourth, we plan to analyze the impact of ubiquitin conjugation on Abh3 and the specific ubiquitination site(s) and characterize the nature of ubiquitin linkage. Fifth, we will show that OTUD4 deubiquitinase activity regulates the cellular stability of Abh3 protein half-life by modulation of proteasome-mediated degradation, and analyze the impact of OTUD4 on Abh3 catalytic activity and on Abh3-mediated alkylation damage repair. Sixth, we will investigate a possible impact of our two enzymes on chemoresistance in our cancer model in the presence of various alkylating agents.Taken together, understanding Abh3 and OTUD4 cooperativity may help us to understand how cellular expression of demethylases/dealkylases is regulated and maintained. Expression of dealkylating enzymes could be a possible explanation how cancer cells mechanistically adopt alkylation damage resistance e.g. during chemotherapy with alkylating agents. The proposed project may allow us to connect DNA alkylation repair to epigenetic events and may promote Abh3 and OTUD4, two oncogene candidates, to potential anticancer targets in the future.