Defining a common mechanism of SAMHD1 function in cancer, HIV, and AGS

定义 SAMHD1 在癌症、HIV 和 AGS 中的常见功能机制

• 批准号: 10480762
• 负责人: Nicole Eileen Bowen
• 金额: $ 4.02万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10480762
• 关键词: ANXA5 gene; Affect; Anti-Retroviral Agents; Apoptosis; Biochemical; Biological Assay; Biology; CD4 Positive T Lymphocytes; Cell Cycle; Cell Cycle Proteins; Cell Cycle Regulation; Cell Differentiation process; Cell Line; Cells; Cellular biology; Cessation of life; Colon Carcinoma; DNA; DNA Double Strand Break; Data; Deoxyribonucleases; Development; Encephalopathies; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Exonuclease; Flow Cytometry; Future; Genes; Genetic Transcription; Growth; HIV; HIV Infections; HIV-1; HIV-2; Immune System Diseases; Immunology; Immunosuppression; Impairment; Individual; Infection; Inherited; Innate Immune Response; Interferons; Interphase Cell; Kinetics; Knock-in; Knock-out; Knowledge; Laboratories; Link; Malignant Neoplasms; Mediating; Metabolism; Mutate; Mutation; NF-kappa B; Normal Cell; Patients; Pharmacologic Substance; Phenotype; Production; Property; Propidium Diiodide; Proteins; Proviruses; RNA-Directed DNA Polymerase; Radiolabeled; Reporting; Research; Rest; Role; SAM Domain; SIV; Sendai virus; Series; Stains; Techniques; Testing; Viral; Viral reservoir; Virus; Virus Latency; Virus Replication; Virus-like particle; Western Blotting; antiretroviral therapy; base; cancer cell; design; disease phenotype; experience; insight; leukemia; macrophage; mutant; novel; novel therapeutics; preservation; pseudotoxoplasmosis syndrome; repaired; tool; tumor molecular fingerprint; virology

Project Summary As of 2019 there were 38.0 million individuals living with HIV globally. This number continues to rise yearly despite advances in HIV treatment. A significant challenge to developing a cure for HIV continues to be the reservoir of latently infected cells. Current antiretrovirals cannot target this subset of non-dividing cells that harbor transcriptionally inactive provirus. It is well established that HIV infection in non-dividing cells has different properties than HIV infection in dividing cells. For example, non-dividing cells harbor dNTP concentrations below the Km of reverse transcriptase which significantly slows the kinetics of viral replication. SAM and HD domain containing protein 1 (SAMHD1) is the host dNTPase responsible for this restriction. A thorough understanding of the contribution of SAMHD1 to the unique environment of HIV-1 infection in non-dividing reservoir cells has the potential to inform novel therapeutics to target this reservoir. To this effect, I propose to characterize a novel series of SAMHD1 mutations that have been implicated in several cancers and use these mutants as tools to further probe the role of SAMHD1 in HIV-1 restriction. I have identified a leukemia and colon cancer associated SAMHD1 mutant, R366C/H, that retains protein stability but loses dNTPase activity. Interactions with cell cycle proteins and involvement in double strand DNA break repair, dNTPase independent functions of SAMHD1, are preserved in this mutant. Interestingly, SAMHD1 mutations that result in impaired dNTPase activity were first reported in Aicardi Goutières Syndrome (AGS), a rare inherited encephalopathy characterized by aberrant type 1 interferon production. Many of these AGS mutants are unable to restrict HIV-1 infection in macrophages due to their inability to deplete cellular dNTPs. Given that the dNTP concentration in cancer cells is 6-11 fold higher than in normal cells and that SAMHD1 knock-out results in cancer cell phenotypes such as increased proliferation and reduced apoptosis, it is likely that the role of SAMHD1 as a dNTPase also involves the enzyme in cancer. Therefore, I hypothesize that the dNTPase deficiency of the R366C/H SAMHD1 cancer mutant contributes to cancer cell phenotypes and abrogates both HIV-1 restriction in non-dividing cells and innate immune response suppression. In Aim 1, I will complete my functional characterization of the R366C/H cancer mutant and determine the impact of these functional deficits on cancer cell phenotypes using biochemical and cell biology techniques. Subsequently in Aim 2, I will use virology, cell biology, and immunology approaches to analyze the effect of the R366C/H cancer mutation on HIV-1 restriction and innate immune suppression, thus using this cancer mutation to probe more traditionally studied SAMHD1 disease phenotypes . Collectively, these complimentary aims will highlight the role of SAMHD1 dNTPase activity in cancer and mechanistically link the involvement of SAMHD1 in cancer, HIV-1 restriction, and AGS.

项目摘要 截至2019年，全球有3800万艾滋病毒感染者。这一数字每年都在继续上升 尽管艾滋病治疗取得了进步。开发治疗艾滋病毒的一个重大挑战仍然是 潜伏感染细胞的储库。目前的抗逆转录病毒药物不能靶向这种非分裂细胞亚群， 转录失活前病毒。众所周知，非分裂细胞中的HIV感染具有不同的 比HIV感染在分裂细胞中的特性。例如，非分裂细胞的dNTP浓度低于 逆转录酶的Km，其显著减慢病毒复制的动力学。SAM和HD域 包含蛋白1（SAMHD 1）的宿主dNTR负责该限制。的透彻理解 SAMHD 1对非分裂储库细胞中HIV-1感染的独特环境的贡献， 有可能为靶向这一储库的新疗法提供信息。为了达到这个效果，我建议把一部小说 一系列SAMHD 1突变与几种癌症有关，并使用这些突变体作为工具， 进一步探讨SAMHD 1在HIV-1限制中的作用。我发现了一种白血病和结肠癌 SAMHD 1突变体R366 C/H，保留蛋白质稳定性但失去dNTR活性。与细胞周期的相互作用 SAMHD 1的蛋白质和参与双链DNA断裂修复的功能， 保存在这个突变体中。有趣的是，导致dNTR活性受损的SAMHD 1突变首先被发现， AGS是一种罕见的遗传性脑病，以异常型为特征， 1干扰素产生。这些AGS突变体中的许多不能限制巨噬细胞中的HIV-1感染， 无法消耗细胞内的dNTPs考虑到癌细胞中的dNTP浓度是癌细胞的6-11倍， 并且SAMHD 1敲除导致癌细胞表型，如增殖增加 和减少的细胞凋亡，SAMHD 1作为dNTR的作用可能也涉及癌症中的酶。 因此，我假设R366 C/H SAMHD 1癌症突变体的dNTR缺陷有助于 对癌细胞表型的抑制，并消除非分裂细胞中的HIV-1限制和先天免疫 反应抑制在目标1中，我将完成R366 C/H癌症突变体的功能表征 并使用生物化学和细胞生物学方法确定这些功能缺陷对癌细胞表型的影响， 生物技术随后在目标2中，我将使用病毒学、细胞生物学和免疫学方法， 分析R366 C/H癌症突变对HIV-1限制和先天免疫抑制的影响，从而 使用这种癌症突变来探测更多传统研究的SAMHD 1疾病表型。总的来说，这些 互补的目的将突出SAMHD 1 dNTR活性在癌症中的作用，并将其与癌症的发生机制联系起来。 SAMHD 1参与癌症、HIV-1限制和AGS。