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

基本信息

批准号：
10254592
负责人：
Nicole Eileen Bowen
金额：
$ 4.6万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10254592
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

项目摘要

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.

项目概要截至 2019 年，全球有 3800 万名艾滋病毒感染者，这一数字还在逐年增加。尽管开发艾滋病毒治疗方法仍面临重大挑战。目前的抗逆转录病毒药物无法针对潜伏感染细胞的这一子集。众所周知，非分裂细胞中的 HIV 感染具有不同的转录活性。例如，非分裂细胞的 dNTP 浓度低于分裂细胞中的 HIV 感染。逆转录酶的 Km 显着减慢病毒复制的动力学。含有蛋白质 1 (SAMHD1) 的宿主 dNTPase 负责这种限制。 SAMHD1 对非分裂储存细胞中 HIV-1 感染的独特环境的贡献为此，我建议描述一种新的治疗方法。一系列与多种癌症有关的 SAMHD1 突变，并使用这些突变体作为工具进一步探讨 SAMHD1 在 HIV-1 限制中的作用我发现了白血病和结肠癌的相关性。 SAMHD1 突变体 R366C/H，保留蛋白质稳定性，但失去与细胞周期的相互作用。蛋白质和参与双链 DNA 断裂修复、SAMHD1 的 dNTPase 独立功能该突变体中首先保留了导致 dNTPase 活性受损的 SAMHD1 突变。据报道，Aicardi Goutières 综合征 (AGS) 是一种以异常类型为特征的罕见遗传性脑病由于许多 AGS 突变体无法限制巨噬细胞中的 HIV-1 感染。鉴于癌细胞中的 dNTP 浓度高出 6-11 倍，因此它们无法耗尽细胞 dNTP。与正常细胞相比，SAMHD1 敲除会导致癌细胞表型，例如增殖增加并减少细胞凋亡，SAMHD1 作为 dNTPase 的作用很可能也涉及癌症中的该酶。因此，我见证了 R366C/H SAMHD1 癌症突变体的 dNTPase 缺陷导致癌细胞表型并消除非分裂细胞中的 HIV-1 限制和先天免疫在目标 1 中，我将完成 R366C/H 癌症突变体的功能表征。并利用生化和细胞分析确定这些功能缺陷对癌细胞表型的影响随后在目标 2 中，我将使用病毒学、细胞生物学和免疫学方法来分析 R366C/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索更传统研究的 SAMHD1 疾病表型。互补的目标将强调 SAMHD1 dNTPase 活性在癌症中的作用，并在机制上将 SAMHD1 与癌症、HIV-1 限制和 AGS 的关系。