IDENTIFICATION OF NOVEL PROTEINS IN TRF1 COMPLEX

TRF1 复合物中新型蛋白质的鉴定

• 批准号: 7355030
• 负责人: Titia de Lange
• 金额: $ 0.15万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355030
• 关键词: IDENTIFICATION; NOVEL; PROTEINS; TRF1; COMPLEX

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Telomeres form a protein-DNA complex that protects chromosome ends from degradation or end-to-end fusion. One of the major telomeric DNA binding proteins in mammals is TRF1, which binds specifically to duplex TTAGGG repeats. TRF1 is thought to interact with tankyrase and TIN2 to regulate telomere length maintenance. Tankyrase is a telomeric poly(ADP-ribose)polymerase (PARP) that binds to the acidic N-terminus of TRF1, and TIN2 is a TRF1-interacting nuclear protein. Using co-immunoprecipitation coupled with mass spectrometry, we attempted to identify novel proteins in the TRF1 complex. A cell line expressing FLAG-HA-tagged TRF1 as well as a cell line containing the empty vector as a control was created in human BJ fibroblast cells that also express hTERT. Using these cells, small-scale FLAG-HA IPs were done to verify that FLAG-HA-TRF1 is interacting with its known binding partners, tankyrase and TIN2, using Western blot analysis and immunofluorescent co-localization experimen ts. The IP was scaled up, and the FLAG-HA-peptide elutions from both cell populations (FLAG-HA-tagged TRF1 and vector control) were run out on a polyacrylamide gel and stained by negative zinc staining. Unique bands seen in the FLAG-HA-TRF1 elution lane were excised from the gel and sent for mass spectrometry analysis. The results of this experiment suggested that TRF1 might be interacting with proteins such as hnRNP-U, hnRNP-D, and nucleolin. Further experiments are being done to investigate the validity of these interactions.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。端粒形成蛋白质-DNA复合物，其保护染色体末端免于降解或端对端融合。哺乳动物中主要的端粒DNA结合蛋白之一是TRF 1，其特异性结合双链TTAGGG重复。TRF 1被认为与端锚聚合酶和TIN 2相互作用以调节端粒长度维持。端锚聚合酶是一种端粒聚（ADP-核糖）聚合酶（PARP），与TRF 1的酸性N-末端结合，TIN 2是一种与TRF 1相互作用的核蛋白。使用免疫共沉淀结合质谱，我们试图确定新的蛋白质在TRF 1复合物。在也表达hTERT的人BJ成纤维细胞中产生表达FLAG-HA标记的TRF 1的细胞系以及含有空载体作为对照的细胞系。使用这些细胞，进行小规模FLAG-HA IP以使用Western印迹分析和免疫荧光共定位实验验证FLAG-HA-TRF 1与其已知结合配偶体端锚聚合酶和TIN 2相互作用。将IP按比例放大，并将来自两个细胞群（FLAG-HA标记的TRF 1和载体对照）的FLAG-HA肽洗脱液在聚丙烯酰胺凝胶上流出，并通过负锌染色进行染色。从凝胶上切下FLAG-HA-TRF 1洗脱泳道中观察到的独特条带，并送去进行质谱分析。本实验的结果表明，TRF 1可能与hnRNP-U，hnRNP-D和核仁素等蛋白质相互作用。正在进行进一步的实验来研究这些相互作用的有效性。