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.

项目摘要该项目旨在开发和应用CASPEX技术来测量细胞的蛋白质变化感染HIV-1。 CASPEX是指催化死亡CAS9（DCAS9）蛋白的组合定位于特定的基因组基因座，融合到工程的抗坏血酸过氧化物酶（APEX2）的位置能够在与融合蛋白接近的球体内生物素化蛋白。 CASPEX启用捕获蛋白质和核酸与与DCAS9复合的引导RNA定义的基因组基因座相邻。这种强大的方法可以与质谱分析相结合，以识别和量化与基因组基因局相关的蛋白质以公正的方式。 HIV-1潜伏期代表开发用于HIV-1治疗和治愈的新疗法的主要障碍。这使用高通量遗传学研究了有关HIV-1潜伏期的分子过程分子筛选和聚焦分子生物学研究。 Caspex有可能提供有关HIV-1潜伏期过程的互补信息，这些信息可以确认先前的工作或开放新途径研究。在这个项目中，我们将开发两种CASPEX方法，并将它们应用于HIV-1潜伏期的模型。在特定目标1中，我们将设计HIV-1潜伏期的细胞系模型，以进行CASPEX分析和表征响应潜伏期逆转的综合HIV-1染色质基因蛋白基因座的蛋白质变化。细胞系中的发现 HIV-1潜伏期的模型并不总是在体内概括，因此在特定目的2中我们将开发一种新颖将CASPEX应用于初级CD4+ T细胞中的方法。成功完成该项目将推进分子对HIV-1潜伏期和潜伏期逆转的理解，有可能告知下一步的发展治疗或治愈HIV-1感染的生成疗法。