Post-translational regulation of the uracil DNA glycosylase

尿嘧啶 DNA 糖基化酶的翻译后调控

• 批准号: 9389793
• 负责人: Brian Patrick Weiser
• 金额: $ 0.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-28 至 2018-08-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9389793
• 关键词: ART protein; Address; Affect; Affinity; Antineoplastic Agents; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Biological Assay; Calorimetry; Catalytic Domain; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cells; Chemicals; Cysteine; Cytosine; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Sequence Alteration; DNA biosynthesis; DNA replication fork; Deamination; Disease; Engineering; Enzymes; Excision; Fluorouracil; Genomic DNA; Genomics; Knock-out; Lesion; Ligase; Ligation; Malignant Neoplasms; Measures; Mentors; Methods; Modification; N-terminal; Nuclear; Pathway interactions; Peptides; Pharmaceutical Preparations; Positioning Attribute; Post-Translational Protein Processing; Post-Translational Regulation; Predisposition; Preparation; Proliferating Cell Nuclear Antigen; Protein Region; Proteins; Recombinant Proteins; Recombinants; Regulation; Role; Site; Structure; Testing; Thymidine; Thymidylate Synthase Inhibitor; Titrations; Toxic effect; Uracil; Uracil Nucleotides; Variant; analog; antigen binding; base; cancer cell; clinical efficacy; cytotoxic; cytotoxicity; ds-DNA; experimental study; fluorescence imaging; insight; milligram; mutant; protein protein interaction; public health relevance; replication factor A; subtiligase; treatment response; uracil-DNA glycosylase

 DESCRIPTION (provided by applicant): Thymidylate synthase inhibitors are a class of drugs that are widely used for the treatment of various cancers. One effect of these compounds is to increase the levels of uracil bases that are incorporated into newly synthesized genomic DNA. Excessive uracil incorporation into DNA can become toxic to cells, and this is believed to contribute to the ability of thymidylate synthase inhibitors to target rapidly dividing cancer cell. The primary DNA repair enzyme that initiates the base excision repair pathway to correct these uracil lesions in genomic DNA is called the nuclear uracil DNA glycosylase (UNG2). We propose a relationship between the uracil excision activity of UNG2 and the efficacy of 5-fluorouracil, a prototypical thymidylate synthase inhibitor. The activity of UNG2 is regulated in part by its localization within the nucleus and its accessibility to sites of DNA damage. Interactions of UNG2 with proliferating cell nuclear antigen (PCNA) and replication protein A (RPA) can affect its localization, specifically to the DNA replication fork; therefore, we reason that its ability to excise uracil from newly synthesized DNA during 5-fluorouracil treatment is als dependent on these protein- protein interactions. Specific UNG2 residues within the PCNA and RPA binding domains can be post- translationally modified, and we hypothesize that these post-translational modifications (PTMs) affect UNG2 interactions with these proteins. Post-translational modification of UNG2 is therefore hypothesized to modulate the susceptibility of cancer cells to 5-fluorouracil toxicity by affecting protein-protein interactions and subsequently removal of uracil from DNA. To test our hypotheses, we will use state-of-the-art protein semi- synthesis methods to generate UNG2 variants that have PTMs incorporated at specific residues. The post- translationally modified UNG2 variants will be used in biochemical assays that quantify their activity and their affinities for PCNA and RPA. The structural basis for modulating UNG2 interactions with PCNA and RPA will also be determined. To confirm that PTMs affect protein-protein interactions and the localization of UNG2 in the nucleus, we will use live cell fluorescence imaging of microinjected semi-synthetic UNG2 variants. Finally, the efficacy of 5-fluorouracil will be examined in cells that are transfected with UNG2 mutants that have altered affinities for PCNA and/or RPA. Together, the results of this interdisciplinary projec will address the contribution of a specific set of UNG2 PTMs towards the efficacy of 5-fluorouracil and other thymidylate synthase inhibitors.

 描述（由申请人提供）：胸苷酸合酶抑制剂是一类广泛用于治疗各种癌症的药物，这些化合物的一个作用是增加掺入新合成的基因组DNA中的尿嘧啶碱基的水平。掺入 DNA 会对细胞产生毒性，这被认为有助于胸苷酸合酶抑制剂靶向快速分裂的癌细胞（启动碱基的主要 DNA 修复酶）。我们提出了 UNG2 的尿嘧啶切除活性与 5-氟尿嘧啶（一种典型的胸苷酸合成酶抑制剂）的功效之间的关系。 UNG2 的部分调节取决于其在细胞核内的定位以及 UNG2 与增殖细胞核抗原的相互作用。 (PCNA) 和复制蛋白 A (RPA) 可以影响其定位，特别是 DNA 复制叉；因此，我们推断其在 5-氟尿嘧啶处理期间从新合成的 DNA 中切除尿嘧啶的能力也依赖于这些蛋白质-蛋白质相互作用。 PCNA 和 RPA 结合域内的特定 UNG2 残基可以进行翻译后修饰，并且我们一直在研究这些翻译后修饰 (PTM) 会影响 UNG2 与这些蛋白质的相互作用。因此，UNG2 率先通过影响蛋白质-蛋白质相互作用以及随后从 DNA 中去除尿嘧啶来调节癌细胞对 5-氟尿嘧啶毒性的敏感性。生成在特定残基处掺入 PTM 的 UNG2 变体的方法 翻译后修饰的 UNG2 变体将用于量化其活性及其对 PCNA 和 RPA 的亲和力的生化测定。还将确定调节 UNG2 与 PCNA 和 RPA 相互作用的基础 为了确认 PTM 影响蛋白质-蛋白质相互作用以及 UNG2 在细胞核中的定位，我们将使用显微注射的半合成 UNG2 的活细胞荧光成像。将在用 UNG2 突变体转染的细胞中检查 5-氟尿嘧啶，这些突变体对 PCNA 和/或 RPA 的亲和力发生改变。 projec 将探讨一组特定的 UNG2 PTM 对 5-氟尿嘧啶和其他胸苷酸合酶抑制剂的功效的贡献。