IDENTIFICATION OF NOVEL PROTEINS IN TRF1 COMPLEX

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。端粒形成一种蛋白质- dna复合物，保护染色体末端免受降解或端到端融合。哺乳动物中主要的端粒DNA结合蛋白之一是TRF1，它特异性地结合双工TTAGGG重复序列。TRF1被认为与坦克酶和TIN2相互作用以调节端粒长度维持。Tankyrase是一种端粒聚（adp -核糖）聚合酶（PARP），与TRF1的酸性n端结合，而TIN2是与TRF1相互作用的核蛋白。使用免疫共沉淀结合质谱法，我们试图鉴定TRF1复合体中的新蛋白。在同样表达hTERT的人BJ成纤维细胞中建立了表达flag - ha标记TRF1的细胞系和包含空载体作为对照的细胞系。利用这些细胞，利用Western blot分析和免疫荧光共定位实验，进行了小规模的FLAG-HA-TRF1的IPs实验，以验证FLAG-HA-TRF1与其已知的结合伙伴tankyrase和TIN2相互作用。放大IP，将两种细胞群（FLAG-HA标记的TRF1和载体对照）的FLAG-HA肽洗脱在聚丙烯酰胺凝胶上进行，并进行负锌染色。在FLAG-HA-TRF1洗脱通道中看到的独特条带从凝胶中切除并送去质谱分析。本实验结果提示TRF1可能与hnRNP-U、hnRNP-D、核仁蛋白等蛋白相互作用。进一步的实验正在进行，以调查这些相互作用的有效性。

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.