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Elucidating the role of TIN2L at the telomere and in dyskeratosis congenita

阐明 TIN2L 在端粒和先天性角化不良中的作用

基本信息

批准号：
8826071
负责人：
NYA D NELSON
金额：
$ 0.88万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8826071
关键词：
Acute Myelocytic Leukemia Affect Age Binding Binding Sites C-terminal Cell Aging Cell Line Cells Chromatin Chromosomes Co-Immunoprecipitations Complex DNA DNA Binding DNA biosynthesis Defect Dependence Dependency Disease Dyskeratosis Congenita Enzymes Functional disorder Gene Mutation General Population Genes Goals Growth Human Human Cell Line Length Malignant Neoplasms Maps Mass Spectrum Analysis Mutate Mutation Nuclear Matrix Nucleoproteins Onset of illness Pancytopenia Pathogenesis Patients Peptides Phenotype Play Population Predisposition Protein Array Protein Binding Protein Fragment Protein Isoforms Proteins RNA Interference Role Structure Subfamily lentivirinae Syndrome TERF1 gene TINF2 gene Telomerase Telomere Length Maintenance Telomere Maintenance Telomere Shortening Telomeric Repeat Binding Protein 2 Testing Time base design high risk intermolecular interaction malignant tongue neoplasm member mutant novel overexpression research study telomere

项目摘要

DESCRIPTION (provided by applicant): Dyskeratosis congenita (DC) is a bone marrow failure and cancer predisposition syndrome. Patients with DC have a particularly high risk of developing acute myeloid leukemia and tongue cancer in comparison to the general population, 200 and 1100 fold respectively. Underlying DC is a defect in the maintenance of telomeres, the essential nucleoprotein structures located at the ends of linear chromosomes. Reflecting this, six of the seven genes mutated in DC encode components or factors required for the assembly or activity of telomerase, the enzyme that replenishes terminal telomeric repeats following DNA replication. Fifteen percent of patients with DC have mutations in the seventh gene, TINF2, which encodes TIN2, a central member of the telomeric binding complex shelterin. The vast majority of TINF2 mutations are de novo, and these patients have shorter telomere lengths and earlier age of disease onset in comparison to other DC patients, underscoring the severe disruption in telomere function conferred by these mutations. Both a short (TIN2S) and a long (TIN2L) isoform of TIN2 are expressed in human cells. TIN2L associates with the telomeres and the nuclear matrix, indicating that it is unlikely to have identical function to TIN2S. All DC-associated TINF2 mutations are heterozygous and map to a region of unclear significance (the DC-cluster), located between its known binding regions and a C- terminal extension unique to TIN2L. The goal of this project is to determine how DC-associated TIN2 mutations result in such severe telomere shortening. We hypothesize that they specifically impact on a unique function of TIN2L, that is absent from TIN2S. This hypothesis is supported by preliminary co-immunoprecipitation studies which showed that TIN2L interacts more robustly with TRF2, a member of the shelterin complex, than TIN2S and that this increased interaction requires the DC-cluster region and a conserved region of TIN2L. We will use mass spectrometry (MS) to identify proteins which bind to TIN2L but not TIN2S, and to determine the role of the DC-cluster in these interactions. To identify the regions of TIN2L necessary for inter/intramolecular interactions we will design an overlapping peptide array containing TIN2L. This array will be probed with TIN2L and TIN2S to identify intramolecular interactions unique to the C terminus and TIN2L with a DC-cluster mutation to determine the dependency of these intramolecular interactions on the DC-cluster. To determine the regions necessary for the increased interaction with TRF2 and binding to the proteins identified via MS, we will probe the array with these proteins. To characterize the role of the two TIN2 isoforms, TRF2, proteins identified via MS and telomerase in the DC phenotype, we will use lentivirus to stably overexpress these proteins in DC patient derived lymphoblastic cell lines, and the effects on telomere length and population doubling time will be determined. Through the study of the wild type function of TIN2S and TIN2L and the role of the DC-cluster in these functions we hope to elucidate the role of TIN2 not only in this devastating telomeric disease, but also in the pathogenesis of cancer in DC patients. This may ultimately have implications for sporadic cancer.

描述（申请人提供）：先天性角化不良（DC）是一种骨髓衰竭和癌症易感综合征。与一般人群相比，DC患者发生急性髓性白血病和舌癌的风险特别高，分别为200和1100倍。潜在的DC是端粒维持的缺陷，端粒是位于线性染色体末端的基本核蛋白结构。反映了这一点，在DC中突变的七个基因中有六个编码端粒酶组装或活性所需的成分或因子，端粒酶是DNA复制后终止末端端粒重复序列的酶。15%的DC患者在第七个基因TINF 2中存在突变，TINF 2编码TIN 2，TIN 2是端粒结合复合物shelterin的中心成员。绝大多数TINF 2突变是新发的，与其他DC患者相比，这些患者具有较短的端粒长度和较早的疾病发作年龄，强调了这些突变所赋予的端粒功能的严重破坏。在人类细胞中表达短型（TIN 2S）和长型（TIN 2L）的TIN 2同种型。TIN 2L与端粒和核基质相关，表明它不太可能具有与TIN 2S相同的功能。所有DC相关的TINF 2突变都是杂合的，并且映射到位于其已知结合区域和TIN 2L特有的C末端延伸之间的意义不明确的区域（DC簇）。该项目的目标是确定DC相关的TIN 2突变如何导致如此严重的端粒缩短。我们假设它们特异性地影响TIN 2L的独特功能，而TIN 2S不存在这种功能。初步的免疫共沉淀研究支持了这一假设，该研究表明，TIN 2L与TRF 2（shelterin复合物的一个成员）的相互作用比TIN 2S更强，并且这种增加的相互作用需要TIN 2L的DC簇区域和保守区域。我们将使用质谱（MS）来识别与TIN 2L结合但不与TIN 2S结合的蛋白质，并确定DC簇在这些相互作用中的作用。为了鉴定分子间/分子内相互作用所必需的TIN 2L区域，我们将设计含有TIN 2L的重叠肽阵列。将用TIN 2L和TIN 2S探测该阵列，以鉴定C末端和具有DC簇突变的TIN 2L特有的分子内相互作用，以确定这些分子内相互作用对DC簇的依赖性。为了确定与TRF 2的相互作用增加以及与通过MS鉴定的蛋白质结合所需的区域，我们将用这些蛋白质探测阵列。为了表征通过MS和端粒酶鉴定的两种TIN 2同种型TRF 2蛋白在DC表型中的作用，我们将使用慢病毒在DC患者来源的淋巴母细胞系中稳定过表达这些蛋白，并确定对端粒长度和群体倍增时间的影响。通过研究TIN 2S和TIN 2L的野生型功能以及DC簇在这些功能中的作用，我们希望阐明TIN 2不仅在这种破坏性端粒疾病中的作用，而且在DC患者的癌症发病机制中的作用。这可能最终会对散发性癌症产生影响。