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

通过脱烷基维持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的，因此烷基化修复对于维持基因组完整性具有重要意义，特别是对于癌症等高增殖组织。烷基化损伤主要由去甲基酶/脱烷基酶直接修复。我们之前的研究结果表明，人类去甲基化酶/脱烷基酶复合物Abh3通过烷基化修复促进癌症进展。tap纯化的前列腺肿瘤细胞中的Abh3复合物显示存在一种去泛素酶，称为卵巢肿瘤结构域OTUD4。引人注目的是，随着时间的推移，用蛋白酶体抑制剂MG132治疗显示Abh3增加，这在对照组中没有观察到。全细胞提取物的Western blot分析表明，Abh3和OTUD4在癌细胞中共表达，shrnai介导的OTUD4敲低与体内（至少在癌细胞中）Abh3表达降低有关。敲除Abh3和OTUD4导致增殖减少以及烷基化损伤敏感性增加。因此，我们的研究结果表明，Abh3的调控可能通过泛素途径进行，这可能是癌症特异性的，Abh3和OTUD4的相互作用可能促进肿瘤进展。这项资助提案围绕着我们的主要假设，即OTUD4通过将其去泛素酶活性与人类Abh3去甲基化酶复合物联系起来促进肿瘤进展。我们首先计划利用肿瘤组织阵列投资Abh3和OTUD4的共定位，并进一步表征Abh3和OTUD4的生化相互作用。其次，我们将在体内阐明OTUD4在DNA烷基化修复和肿瘤生长中的功能。第三，我们将研究OTUD4在体外和体内是否使Abh3去泛素化，并表征OTUD4去泛素酶活性的性质。第四，我们计划分析泛素结合对Abh3和特定泛素化位点的影响，并表征泛素连接的性质。第五，我们将证明OTUD4去泛素酶活性通过调节蛋白酶体介导的降解来调节Abh3蛋白半衰期的细胞稳定性，并分析OTUD4对Abh3催化活性和Abh3介导的烷基化损伤修复的影响。第六，我们将研究在各种烷基化剂存在的情况下，我们的癌症模型中两种酶对化学耐药的可能影响。综上所述，了解Abh3和OTUD4的协同作用可能有助于我们了解去甲基化酶/脱烷基酶的细胞表达是如何被调节和维持的。脱烷基酶的表达可能解释了癌细胞如何机械地采用烷基化损伤抗性，例如在烷基化剂化疗期间。该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，并可能在未来促进Abh3和OTUD4这两个致癌基因候选基因成为潜在的抗癌靶点。

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.