Characterizing chromatin protein dynamics in HIV-1 latency with a CASPEX approach

使用 CASPEX 方法表征 HIV-1 潜伏期染色质蛋白动态

基本信息

批准号：
10547359
负责人：
Jeffrey R Johnson
金额：
$ 21.13万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-08 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10547359
关键词：
Address Antibodies Biology Biotin Biotinylation CD4 Positive T Lymphocytes Cell Line Cell model Cells Chimeric Proteins Chromatin Chronic Communities Complement Complex DNA DNA-Protein Interaction Development Engineering Enzymes Genetic Genome Genomic Segment Guide RNA HIV HIV-1 Hydrogen Peroxide Infection Inflammation Lead Long Terminal Repeats Mass Spectrum Analysis Measures Methods Modeling Molecular Molecular Biology Nucleic Acids Peroxidases Persons Phenols Physiologic pulse Process Protein Dynamics Proteins Proteomics Recombinants Research Research Personnel Ribonucleoproteins Sampling Shock System T-Lymphocyte Technology Therapeutic Virus Virus Latency Western Blotting Work Yellow fever virus antiretroviral therapy ascorbate chromatin immunoprecipitation chromatin protein comorbidity endonuclease genomic locus improved in vivo mutant new technology next generation novel strategies novel therapeutics phenoxy radical prevent reactivation from latency recruit response screening small molecule therapeutic development therapy development viral rebound virus host interaction vpr Gene Products

项目摘要

PROJECT SUMMARY This project aims to develop and apply CASPEX technologies to measure protein changes in cells latently infected with HIV-1. CASPEX refers to the combination of a catalytically-dead Cas9 (dCas9) protein that can localize to a specific genomic locus fused to an engineered ascorbate peroxidase enzyme (APEX2) that is capable of biotinylating proteins within a sphere of proximity to the fusion protein. CASPEX enables the capture of proteins and nucleic acids in proximity to a genomic locus defined by guide RNAs complexed with dCas9. This powerful approach can be combined with mass spectrometry analysis to identify and quantify changes in proteins associated with genomic loci in an unbiased manner. HIV-1 latency represents the major barrier to developing new therapies for HIV-1 treatment and cure. The molecular processes governing HIV-1 latency have been investigated using high-throughput genetics, small molecule screening, and focused molecular biology studies. CASPEX has the potential to provide complementary information regarding HIV-1 latency processes that can confirm prior work or open new avenues of research. In this project, we will develop two CASPEX approaches and apply them to models of HIV-1 latency. In Specific Aim 1, we will engineer cell line models of HIV-1 latency for CASPEX analysis and characterize protein changes at integrated HIV-1 chromatin loci in response to latency reversal. Findings made in cell line models of HIV-1 latency are not always recapitulated in vivo, so in Specific Aim 2 we will develop a novel approach to apply CASPEX in primary CD4+ T cells. Successful completion of this project will advance the molecular understanding of HIV-1 latency and latency reversal, potentially informing on the development of next- generation therapies to treat or cure HIV-1 infection.

项目概要该项目旨在开发和应用CASPEX技术来测量细胞中潜在的蛋白质变化感染 HIV-1。 CASPEX 是指催化死亡 Cas9 (dCas9) 蛋白的组合，可以定位于与工程抗坏血酸过氧化物酶 (APEX2) 融合的特定基因组位点，该酶是能够对融合蛋白附近范围内的蛋白质进行生物素化。 CASPEX 启用捕获靠近由与 dCas9 复合的指导 RNA 定义的基因组位点的蛋白质和核酸。这种强大的方法可以与质谱分析相结合，以识别和量化以公正的方式与基因组位点相关的蛋白质。 HIV-1 潜伏期是开发 HIV-1 治疗和治愈新疗法的主要障碍。这控制 HIV-1 潜伏期的分子过程已经使用高通量遗传学、小分子筛选和重点分子生物学研究。 CASPEX 有潜力提供有关 HIV-1 潜伏过程的补充信息，可以确认先前的工作或开辟新的途径研究。在这个项目中，我们将开发两种 CASPEX 方法并将其应用于 HIV-1 潜伏期模型。在具体目标 1 中，我们将设计 HIV-1 潜伏期的细胞系模型，用于 CASPEX 分析并表征 HIV-1 染色质整合位点的蛋白质变化响应潜伏期逆转。细胞系中的发现 HIV-1 潜伏期模型并不总是在体内重现，因此在特定目标 2 中，我们将开发一种新的在原代 CD4+ T 细胞中应用 CASPEX 的方法。该项目的顺利完成将推动对 HIV-1 潜伏期和潜伏期逆转的分子理解，可能为下一代的发展提供信息治疗或治愈 HIV-1 感染的新一代疗法。