Elucidating the Chemistry and Biology of Nucleic Acid Cytidine Deaminases in HIV

阐明 HIV 核酸胞苷脱氨酶的化学和生物学

• 批准号: 8136827
• 负责人: Rahul Manu Kohli
• 金额: $ 13.2万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136827
• 关键词: Biology; Chemistry; Cytidine Deaminase; HIV; Nucleic Acids

The primary investigator is an MD/PhD trained infectious diseases physician with an interest in understanding enzymes that generate diversity in host-pathogen interactions. In the proposed work, the PI aims to bring his prior experience in enzyme mechanisms and develop new training through virologic experiments and immunologic studies. A remarkable group of enzymes, the polynucleotide cytidine deaminases of the AID/APOBEC family, play both constructive and destructive roles in struggle against HIV. On one hand, deamination by the family member APOBEC3G interferes with the integrity of the pathogen genome. In turn, HIV has evolved the lentiviral protein Vif as an evasive means to counteract human APOBEC3G. Infection with HIV is also associated with immune activation, which can result in increased expression of a B-cell specific deaminase family member, AID. AID physiologically serves as the chief catalyst governing antibody diversity through the introduction of targeted uracil lesions in antibody variable genes or switch regions which ultimately result in higher affinity antibodies of altered isotype. Aberrant regulation and expression of AID has increasingly been associated with Non-Hodgkins lymphoma, the leading AIDS-defining malignancy in HIV infected patients. Despite the importance of these cytidine deaminases, little is know about the nature of their interaction with their nucleic acid targets. This proposal addresses the hypothesis that the molecular interactions that lead to catalysis and binding of nucleic acids are critical determinants of their proper physiologic function. The studies aim to decipher and perturb these molecular interactions. Initially, structure-based hypotheses will be used to localize the protein determinants of sequence preference and resolve the mode of binding to the nucleic acid backbone. By utilizing novel loop graft mutant enzymes with altered sequence preference, the impact of perturbed sequence specificity on retroviral restriction (APOBEC3G) or antibody diversity and chromosomal translocations (AID) will be explored. To understand catalysis by AID/APOBEC enzymes, nucleoside analogs will be introduced into oligonucleotides via chemical or chemoenzymatic methods and are used to characterize the kinetics of deamination and the inhibition of pro-oncogenic AID activity. Taken together, a full characterization of the AID/APOBEC-nucleic acid complex ¿ binding and catalysis ¿ will provide a molecular basis for the action of this important enzyme family in vitro and in vivo. Through mentored training, the PI will develop the broad based research skills necessary to examine biological and biochemical aspects of diversity generation in host-pathogen interactions upon an ultimate transition to independence.

主要研究者是经过医学博士/博士培训的传染病医生，其兴趣是 了解在宿主-病原体相互作用中产生多样性的酶。在拟议的工作中，PI 目的是把他以前在酶机制方面的经验和通过病毒学开发新的培训 实验和免疫学研究。 一组引人注目的酶，AID/APOBEC家族的多核苷酸胞苷脱氨酶， 在与艾滋病毒的斗争中发挥建设性和破坏性的作用。一方面，家庭的脱氨作用 成员APOBEC 3G干扰病原体基因组的完整性。反过来，艾滋病毒已经进化出 慢病毒蛋白Vif作为对抗人APOBEC 3G的逃避手段。感染艾滋病毒也是 与免疫激活相关，这可能导致B细胞特异性脱氨酶表达增加 家人，援助。AID在生理学上作为主要的催化剂，通过免疫调节调节抗体多样性。 在抗体可变基因或转换区中引入靶向尿嘧啶损伤， 改变同种型的更高亲和力抗体。艾滋病的异常调节和表达越来越受到人们的关注。 与非霍奇金淋巴瘤相关，非霍奇金淋巴瘤是HIV感染患者中主要的AIDS定义恶性肿瘤。 尽管这些胞苷脱氨酶的重要性，很少有人知道他们的相互作用的性质， 它们的核酸靶标。这一提议提出了一个假设，即导致 核酸的催化和结合是其适当生理功能的关键决定因素。研究 旨在破译和扰乱这些分子间的相互作用。最初，基于结构的假设将用于 定位序列偏好的蛋白质决定簇并解析与核酸的结合模式 骨干通过利用具有改变的序列偏好性的新型环移植突变体酶， 逆转录病毒限制性酶切干扰序列特异性（APOBEC 3G）或抗体多样性和染色体 我们将研究易位（AID）。了解AID/APOBEC酶的催化作用，核苷类似物 将通过化学或化学酶法引入寡核苷酸中，并用于表征 脱氨动力学和抑制原癌AID活性。加在一起， AID/APOBEC-核酸复合物结合和催化的表征将提供一种分子 这一重要的酶家族在体外和体内的行动的基础。通过指导培训，PI将 发展必要的基础广泛的研究技能，以研究多样性的生物和生物化学方面 在宿主-病原体相互作用中产生，最终过渡到独立。