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

主要研究人员是一名医学博士/博士学位的传染病内科医生，对 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 旨在利用他在酶机制方面的先前经验，并通过病毒学开发新的培训 实验和免疫学研究。 一组值得注意的酶，AID/APOBEC家族的多核苷酸胞苷脱氨酶， 在抗击艾滋病毒的斗争中发挥建设性和破坏性作用。一方面，家庭的欺凌 APOBEC3G成员干扰病原体基因组的完整性。反过来，艾滋病毒进化成了 慢病毒蛋白Vif作为一种逃避手段来对抗人类APOBEC3G。感染艾滋病毒也是 与免疫激活有关，可导致B细胞特异性脱氨酶表达增加 家庭成员，艾滋病。从生理上讲，AID是控制抗体多样性的主要催化剂，通过 在抗体可变区或转换区引入靶向尿嘧啶损伤，最终导致 同型改变的高亲和力抗体。艾滋病的异常调控和表达越来越受到人们的关注 与非霍奇金淋巴瘤有关，非霍奇金淋巴瘤是艾滋病毒感染患者中定义艾滋病的主要恶性肿瘤。 尽管这些胞苷脱氨酶很重要，但人们对它们与之相互作用的性质知之甚少 它们的核酸靶标。这一建议解决了这样的假设，即导致 核酸的催化和结合是其正常生理功能的关键决定因素。这些研究 目的是破译和扰乱这些分子相互作用。最初，基于结构的假设将被用于 定位序列偏好的蛋白质决定因素并解析与核酸结合的模式 我的脊梁。通过利用具有改变的序列偏好的新型环状嫁接突变酶， 受干扰的逆转录病毒限制性内切酶序列特异性(APOBEC3G)或抗体多样性和染色体 本课程将探讨易位(AID)。了解AID/APOBEC酶、核苷类似物的催化作用 将通过化学或化学酶方法引入到寡核苷酸中，并用于表征 脱氨动力学和抑制致癌活性。加在一起，一个完整的 对AID/APOBEC-核酸复合体的结合和催化的表征将提供一种分子 为这个重要的酶家族在体外和体内的作用奠定了基础。通过辅导培训，PI将 发展必要的基础广泛的研究技能，以考察多样性的生物和生化方面 在最终过渡到独立时，宿主-病原体相互作用的产生。

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.