THE MOLECULAR MECHANISMS OF ADULT T CELL LEUKEMIA INDUCTION BY HTLV-1 TAX

HTLV-1 TAX 诱导成人 T 细胞白血病的分子机制

• 批准号: 8360374
• 负责人: MAUREEN SHUH
• 金额: $ 7.34万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360374
• 关键词: Adult; Amino Acids; Ammonium Sulfate; Area; Binding; Biochemical; Biological Assay; Biomedical Research; Blood Donations; CD4 Positive T Lymphocytes; Caribbean region; Cells; Central Africa; Characteristics; Chromatography; Circular Dichroism Spectroscopy; Complex; DNA; DNA Binding; Data; Fractionation; Funding; Genetic Transcription; Grant; Human; Human T-lymphotropic virus 1; Infection; Italy; Japan; Laboratories; Length; Louisiana; Malignant Neoplasms; Methods; Molecular; National Center for Research Resources; Oncogenic; Patients; Phenotype; Precipitation; Principal Investigator; Process; Production; Property; Proteins; Recovery; Regulation; Research; Research Infrastructure; Resources; Serum Response Factor; Source; Structure; T-Cell Leukemia; T-Lymphocyte; Taxes; United States; United States National Institutes of Health; Vaccines; Vascular blood supply; Viral Proteins; Virus; cost; monomer; mutant; outcome forecast; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Background. Human T cell leukemia virus type 1 (HTLV-1) is causes a human cancer known as adult T cell leukemia (ATL). Approximately 11-20 million people are infected (HTLV-1+) in the world with pockets of infection occurring in the Caribbean, central Africa, Japan, and Italy. United States blood donations are routinely assayed for the presence of HTLV-1, and about 0.025% of the U.S. blood supply is positive for the virus. There is no vaccine for the virus. The virus infects and integrates its proviral DNA in human CD4+ T cells, and after a latency period, about 3-5% of HTLV-1+ patients develop ATL, a debilitating and aggressive cancer with no known cure and a poor prognosis. Rationale and Hypothesis. The research in the laboratory is focused on the viral protein Tax, a 40 kDa, 353 amino acid, phosphorylated protein. Numerous studies since the early 1980s have demonstrated that Tax is the oncogenic protein of HTLV-1, and introduction of tax into normal human T cells results in transformation. The mechanism by which Tax initiates and maintains the transformed phenotype of the host cell is not yet known. One factor that has impeded the progress in this area is the lack of structural data (secondary and tertiary structure) and other physical characteristics for Tax. The main hypothesis of our research is that Tax activation of serum-response-factor (SRF)-dependent transcription leads to the initiation of HTLV-1 transformation. The focused hypothesis of the proposed project is that Tax regulates the binding activities of SRF and its partner ternary complex factor (TCF) -- which bind the DNA, forming a Tax/SRF/TCF/DNA complex  which results in a higher Ka of the complex with Tax than without Tax. Furthermore, we believe that Tax regulation of the complex is directly dependent on its structure. Methods. The focus of the project is to characterize the biophysical properties of Tax alone and as part of the SRF/DNA complex. The methods we use are (1.) to express Tax, SRF, and TCF in bacterial and human cells, (2.) to purify the proteins by a two-step process of double ammonium sulfate precipitation, followed by size fractionation chromatography, (3.) to determine the binding interactions of wild-type and mutant Tax with and without the SRF/TCF/DNA complexes, and (4.) to use circular dichroism spectroscopy to determine the secondary structure of wild-type and mutant Tax with and without the SRF/TCF/DNA complexes. Results. We have been able to express and purify Tax as a monomer which is important for the biochemical studies. We have obtained SRF as well although we are unable to separate the full-length 67 kDa SRF from the truncated SRF which initiates from a second AUG downstream of the first AUG. Discussion and Interpretation. Our percent recovery is low and we will need to increase our production of the proteins in order to begin the biochemical experiments.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 背景人类T细胞白血病病毒1型（HTLV-1）是一种导致人类癌症的病毒，称为成人T细胞白血病（ATL）。全世界约有1100万至2000万人感染（HTLV-1+），在加勒比海、中非、日本和意大利发生感染。美国捐赠的血液会定期检测是否存在HTLV-1，大约0.025%的美国血液供应对该病毒呈阳性。没有针对该病毒的疫苗。该病毒感染并将其前病毒DNA整合到人CD 4 + T细胞中，经过一段潜伏期后，约3-5%的HTLV-1+患者发展为ATL，这是一种衰弱和侵袭性癌症，没有已知的治愈方法，预后不良。 基本原理和假设。实验室的研究集中在病毒蛋白Tax上，Tax是一种40 kDa，353个氨基酸的磷酸化蛋白。自20世纪80年代初以来的大量研究表明，Tax是HTLV-1的致癌蛋白，将Tax引入正常人T细胞会导致转化。Tax启动和维持宿主细胞转化表型的机制尚不清楚。阻碍这一领域取得进展的一个因素是缺乏结构数据（二级和三级结构）和税收的其他物理特征。我们研究的主要假设是Tax激活血清反应因子（SRF）依赖性转录导致HTLV-1转化的启动。该项目的重点假设是Tax调节SRF及其伴侣三元复合物因子（TCF）的结合活性-它们结合DNA，形成Tax/SRF/TCF/DNA复合物  这导致有税收的络合物比没有税收的络合物具有更高的Ka。此外，我们认为，综合体的税收监管直接取决于其结构。 方法.该项目的重点是确定Tax本身和作为SRF/DNA复合物一部分的生物物理特性。我们使用的方法是（1）。在细菌和人类细胞中表达Tax、SRF和TCF，（2.）通过两步硫酸铵沉淀法和随后的大小分级色谱法纯化蛋白质，（3.）确定野生型和突变型Tax与和不与SRF/TCF/DNA复合物的结合相互作用，和（4.）使用圆二色光谱法测定野生型和突变型Tax在有和没有SRF/TCF/DNA复合物的情况下的二级结构。 结果我们已经能够表达和纯化Tax作为一个单体，这是重要的生化研究。我们也获得了SRF，尽管我们不能从截短的SRF中分离全长67 kDa SRF，截短的SRF起始于第一个AUG下游的第二个AUG。 讨论和解释。我们的回收率很低，我们需要增加蛋白质的产量，以便开始生化实验。