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

描述（由申请人提供）：主要研究者是一名MD/PhD培训的感染性疾病医生，对了解在宿主-病原体相互作用中产生多样性的酶感兴趣。在拟议的工作中，PI的目的是通过病毒学实验和免疫学研究，带来他在酶机制方面的经验，并开发新的培训。一组引人注目的酶，AID/APOBEC家族的多核苷酸胞苷脱氨酶，在对抗HIV的斗争中发挥建设性和破坏性作用。一方面，通过家族成员APOBEC 3G的脱氨基作用干扰病原体基因组的完整性。反过来，HIV已经进化出慢病毒蛋白Vif作为对抗人类APOBEC 3G的逃避手段。艾滋病毒感染还与免疫激活有关，这可能导致B细胞特异性脱氨酶家族成员AID的表达增加。AID在生理学上作为控制抗体多样性的主要催化剂，其通过在抗体可变基因或开关区中引入靶向尿嘧啶损伤而最终导致改变同种型的更高亲和力抗体。AID的异常调节和表达越来越多地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与他们的核酸目标的性质。这一提议提出了这样的假设，即导致核酸催化和结合的分子相互作用是其适当生理功能的关键决定因素。这些研究旨在破译和干扰这些分子相互作用。最初，基于结构的假设将用于定位序列偏好的蛋白质决定簇，并解析与核酸骨架结合的模式。通过利用具有改变的序列偏好的新型环移植突变体酶，将探索干扰的序列特异性对逆转录病毒限制（APOBEC 3G）或抗体多样性和染色体易位（AID）的影响。为了理解AID/APOBEC酶的催化作用，将通过化学或化学酶促方法将核苷类似物引入寡核苷酸中，并用于表征脱氨基的动力学和原癌AID活性的抑制。总之，AID/APOBEC-核酸复合物的完整表征-结合和催化-将为该重要酶家族在体外和体内的作用提供分子基础。通过指导培训，PI将发展必要的基础广泛的研究技能，以检查生物和生物化学方面的多样性产生在宿主-病原体相互作用后，最终过渡到独立。 相关性免疫系统使用酶催化胞嘧啶的靶向脱氨基作用来限制HIV并产生高亲和力抗体，尽管相同的机制可能是促癌的。该提案旨在通过生物化学方法定义和生物学干扰分子相互作用，使AID/APOBEC家族的多核苷酸胞苷脱氨酶能够保护免受感染。

DESCRIPTION (provided by applicant): 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 known 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. RELEVANCE The immune system uses enzymes that catalyze the targeted deamination of cytosine to restrict HIV and make high affinity antibodies, though the same mechanisms can potentially be pro-oncogenic. This proposal aims to biochemically define and biologically perturb the molecular interactions that allow polynucleotide cytidine deaminase enzymes of the AID/APOBEC family to protect against infection.