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

主要研究者是一位受过医学博士/博士学位训练的传染病医生，对以下领域感兴趣 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 旨在利用他之前在酶机制方面的经验，并通过病毒学开展新的培训 实验和免疫学研究。 AID/APOBEC 家族的多核苷酸胞苷脱氨酶是一组非凡的酶， 在抗击艾滋病毒的斗争中既发挥建设性作用，也发挥破坏性作用。一方面，家族的脱氨 APOBEC3G 成员干扰病原体基因组的完整性。反过来，艾滋病毒又进化出了 慢病毒蛋白 Vif 作为对抗人类 APOBEC3G 的规避手段。感染艾滋病毒也 与免疫激活相关，可导致 B 细胞特异性脱氨酶表达增加 家庭成员，援助。 AID 在生理上是通过以下方式控制抗体多样性的主要催化剂： 在抗体可变基因或开关区域中引入靶向尿嘧啶损伤，最终导致 改变同种型的亲和力更高的抗体。 AID 的异常调节和表达已日益受到关注 与非霍奇金淋巴瘤有关，非霍奇金淋巴瘤是艾滋病毒感染者中主要的艾滋病定义恶性肿瘤。 尽管这些胞苷脱氨酶很重要，但人们对它们与胞苷脱氨酶相互作用的性质知之甚少。 他们的核酸目标。该提议提出了这样的假设：分子相互作用导致 核酸的催化和结合是其正常生理功能的关键决定因素。研究 旨在破译和扰乱这些分子相互作用。最初，基于结构的假设将用于 定位序列偏好的蛋白质决定因素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的新型环移植突变酶， 逆转录病毒限制 (APOBEC3G) 或抗体多样性和染色体的序列特异性受到干扰 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寡核苷酸并用于表征 脱氨基动力学和促癌 AID 活性的抑制。综合起来，就是一个完整的 AID/APOBEC-核酸复合物的表征“结合和催化”将提供分子 这一重要酶家族在体外和体内作用的基础。通过指导培训，PI 将 培养检查多样性的生物和生化方面所需的广泛研究技能 最终过渡到独立时宿主与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.