Delineating a New Mechanism of Foreign DNA Restriction in Human Cells

描绘人类细胞中外源 DNA 限制的新机制

• 批准号: 8132470
• 负责人: Michael Allen Carpenter
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-06 至 2013-01-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8132470
• 关键词: Bacteria; Biochemical; Cells; Complex; Cytoplasm; Cytosine; Cytosine deaminase; DNA; DNA Viruses; Deamination; Endosomes; Engineered Gene; Enzymes; Family member; Fractionation; Genetic Engineering; Human; Immune response; Interferons; Lead; Mediating; Modeling; Molecular; Mutation; Nature; Outcome; Phagocytes; Process; Proteins; Safety; Single-Stranded DNA; System; Testing; Therapeutic; Transfection; Uracil; combat; ds-DNA; human disease; inhibitor/antagonist; non-viral gene therapy; novel; plasmid DNA; prevent; protective effect; public health relevance; research study; response; restriction enzyme; small molecule; transition mutation; uptake

DESCRIPTION (provided by applicant): The APOBEC3 DNA cytosine deaminases are a fundamental part of the mammalian innate immune response. In particular, APOBEC3A mediates foreign DNA restriction in human cells with an outcome analogous to the protective effects of restriction enzymes in bacteria. This enzyme's expression levels are dramatically up-regulated by interferon, CpG oligos, transfected double-stranded DNA, and some DNA viruses. Foreign DNA recovered from APOBEC3A-expressing cells contains enormous levels of cytosine deamination with as many as two thirds of the cytosines converted to uracils (detected as C/G to T/A transition mutations). First, we will use model DNA substrates and biochemical approaches to test the hypothesis that APOBEC3A prefers double-stranded DNA substrates, as opposed to other family members that recognize single-stranded DNA. Second, subcellular localization and biochemical fractionation experiments will be used to test the hypothesis that chromosomal DNA is not targeted by APOBEC3A because catalytically active enzyme is localized exclusively to the cytoplasm or a cytoplasmic sub-compartment such as the endosomes. Finally, we propose to advance our preliminary studies on novel small molecules to identify those that specifically inhibit APOBEC3A activity in human cells. We anticipate that bona fide APOBEC3A inhibitors will make human cells more amendable to genetic engineering. Overall, we anticipate elucidating more of the APOBEC3A-mediated foreign DNA restriction mechanism and discovering lead compounds to transiently neutralize this innate defense and render cells more susceptible to genetic engineering. PUBLIC HEALTH RELEVANCE: Foreign DNA poses an intrinsic threat to cells and the human APOBEC3 proteins prevent its uptake. A molecular understanding of this process will have implications for combating human disease through enhancing innate immune responses and, in some instances, through diminishing the innate responses to enhance the uptake of therapeutic DNA.

描述（由申请方提供）：APOBEC 3 DNA胞嘧啶脱氨酶是哺乳动物先天免疫应答的基本部分。特别是，APOBEC 3A介导人类细胞中的外源DNA限制，其结果类似于细菌中限制性酶的保护作用。该酶的表达水平受到干扰素、CpG oligos、转染的双链DNA和一些DNA病毒的显着上调。从表达APOBEC 3A的细胞中回收的外源DNA含有大量的胞嘧啶脱氨基作用，其中多达三分之二的胞嘧啶转化为尿嘧啶（检测为C/G至T/A转换突变）。首先，我们将使用模型DNA底物和生物化学方法来测试APOBEC 3A偏好双链DNA底物的假设，而不是识别单链DNA的其他家族成员。其次，亚细胞定位和生物化学分级分离实验将用于检验染色体DNA不被APOBEC 3A靶向的假设，因为催化活性酶仅定位于细胞质或细胞质亚区室如内体。最后，我们建议推进我们对新型小分子的初步研究，以确定那些特异性抑制人类细胞中APOBEC 3A活性的小分子。我们预计真正的APOBEC 3A抑制剂将使人类细胞更适合基因工程。总的来说，我们预计将阐明更多的APOBEC 3A介导的外源DNA限制机制，并发现先导化合物来暂时中和这种先天防御，使细胞更容易受到基因工程的影响。 公共卫生相关性：外来DNA对细胞构成内在威胁，人类APOBEC 3蛋白阻止其吸收。对这一过程的分子理解将对通过增强先天免疫应答以及在某些情况下通过减少先天应答以增强治疗性DNA的摄取来对抗人类疾病产生影响。