Regulation of the Mono-Ubiquitination of the Fanconi Anemia D2 Protein

描述（由申请人提供）：范可尼贫血（Fanconi anemia， FA）是一种罕见的遗传性疾病，以儿童骨髓衰竭和先天性异常为特征。在获得性骨髓衰竭的病例中，FA基因也经常发生突变。FA蛋白在FA- BRCA通路中与肿瘤抑制蛋白BRCA1和BRCA2 （FANCD1）协同作用，修复受损的DNA并阻止细胞转化。FA-BRCA通路激活的一个关键步骤是FANCD2和FANCI蛋白的单泛素化。重要的是，FANCD2和FANCI单泛素化的细胞调控仍然知之甚少。此外，单泛素化的FANCD2和FANCI在DNA损伤反应中的生理功能在很大程度上仍然未知。本研究计划的主要目标是系统地解决这些关键的未解问题。为了实现这一目标，我们最近确定p21周期蛋白依赖性激酶抑制剂在DNA损伤诱导的FANCD2单泛素化调控中起重要作用。此外，利用计算机生物信息学方法，我们在FANCD2中鉴定了一个假定的泛素结合域（UBD）和一个近端泛素样域（UbL）。初步实验证实了FANCD2与泛素之间的非共价相互作用。在本提案中，我们计划系统地表征p21以及假定的UBD和UbL结构域在FANCD2单泛素化和FA-BRCA通路激活中的作用。由于FANCD2和FANCI单泛素化缺陷是大约90% FA患者以及一般（非FA）人群中一部分骨髓发育不全病例的细胞特征，因此对这种翻译后修饰的调控和功能的研究将有助于更好地理解骨髓的维持和稳定性。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a rare genetic disease characterized by pediatric bone marrow failure and congenital abnormalities. The FA genes are also frequently mutated in cases of acquired bone marrow failure. The FA proteins function cooperatively with the tumor suppressor proteins BRCA1 and BRCA2 (FANCD1) in the FA- BRCA pathway to repair damaged DNA and to prevent cellular transformation. A critical step in the activation of the FA-BRCA pathway is the mono-ubiquitinaton of the FANCD2 and FANCI proteins. Importantly, the cellular regulation of FANCD2 and FANCI mono-ubiquitination remains poorly understood. Furthermore, the physiological function of mono-ubiquitinated FANCD2 and FANCI in the DNA damage response remains largely unknown. The major goal of this research proposal is to systematically address these critical unanswered questions. Towards this goal, we have recently determined that the p21 cyclin dependent kinase inhibitor plays an important role in the regulation of DNA damage-inducible FANCD2 mono-ubiquitination. Furthermore, using in silico bioinformatic approaches, we have identified a putative ubiquitin-binding domain (UBD) and a proximal ubiquitin-like domain (UbL) in FANCD2. Preliminary experiments have confirmed a non- covalent interaction between FANCD2 and ubiquitin. In this proposal, we plan to systematically characterize the role of p21, as well as the putative UBD and UbL domains, in the mono-ubiquitination of FANCD2 and the activation of the FA-BRCA pathway. As defective FANCD2 and FANCI mono-ubiquitination is a cellular feature of >90% of FA patients as well as a subset of cases of marrow aplasia in the general (non-FA) population, the study of the regulation and function of this post-translational modification stands to impart a greater understanding of bone marrow maintenance and stability in general.