IDENTIFICATION OF CSE4-INTERACTING PROTEINS

CSE4 相互作用蛋白的鉴定

• 批准号: 8171384
• 负责人: Susan Biggins
• 金额: $ 0.14万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171384
• 关键词: Aneuploidy; Anions; Arginine; Cell Cycle; Centromere; Chromatin Structure; Chromatography; Chromosomes; Computer Retrieval of Information on Scientific Projects Database; Congenital Abnormality; DNA; DNA Sequence; Data; Deposition; Epigenetic Process; Funding; Genomic Instability; Grant; Histone H3; Immunoprecipitation; Institution; Kinetochores; Lead; Lysine; Mass Spectrum Analysis; Mediating; Mutate; Nucleosomes; Proteins; Proteolysis; Research; Research Personnel; Resources; Saccharomycetales; Site; Source; Specific qualifier value; Testing; Ubiquitin-mediated Proteolysis Pathway; United States National Institutes of Health; Variant; Yeasts; daughter cell; prevent; protein structure; tumor

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. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of all tumors and many birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA sequences and mediate attachment to the spindle. One hallmark of all eukaryotic kinetochores is an essential centromeric histone H3 (CenH3) variant that localizes exclusively to centromeres and replaces canonical histone H3 in centromeric nucleosomes. Because centromeric DNA sequences are not conserved, CenH3 has been proposed to be the epigenetic component that specifies the site of kinetochore assembly. Although CenH3 is an essential component of all kinetochores and is required for centromeric chromatin structure, little is known about CenH3 incorporation into centromeric DNA. We therefore propose to purify the budding yeast CenH3, Cse4, to identify interacting proteins that may regulate its exclusive deposition at the centromere. Because we previously found that Cse4 is degraded by ubiquitin-mediated proteolysis, we created a stable Cse4 protein by mutating all of the lysine residues to arginine (Cse4K16R) (Collins et al., 2004). The Cse4K16R protein is significantly stabilized allowing the accumulation of soluble Cse4 protein. Because Cse4K16R is still targeted to the centromere, it still interacts with the deposition factors that load it at the centromere. We have therefore constructed a Cse4K16R-FLAG protein and purified it by anti-FLAG immunoprecipitation (data not shown). We are currently performing a second step purification using anion exchange chromatography and propose to submit this purified protein for mass spectrometry to identify interacting proteins. We will subsequently test whether the proteins identified interact with the wildtype Cse4 protein and assist in its loading at the yeast centromere. Taken together, these studies should identify proteins that lead to Cse4 deposition at the centromere. Collins, K. A., S. Furuyama and S. Biggins. 2004. Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol. 14 (21) 1968-72.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在每个细胞周期中，染色体必须准确地分割到子细胞，以防止基因组不稳定和非整倍体，这是所有肿瘤和许多出生缺陷的标志。染色体使用着丝点进行分离，着丝点是一种特殊的蛋白质结构，组装在着丝粒DNA序列上，并中介附着到纺锤体上。所有真核着丝点的标志之一是一个重要的着丝粒的组蛋白H3(CenH3)变异体，它专门定位于着丝粒，并取代着丝粒核小体中的典型的组蛋白H3。由于着丝粒DNA序列不保守，CenH3被认为是决定着丝粒组装位置的表观遗传学成分。虽然CenH3是所有着丝点的重要组成部分，也是着丝粒染色质结构所必需的，但对CenH3掺入着丝粒DNA的情况知之甚少。因此，我们建议对发芽酵母CenH3、Cse4进行纯化，以确定可能调节其在着丝粒独占沉积的相互作用蛋白。 由于我们先前发现Cse4可通过泛素介导的蛋白质降解，因此我们通过将所有赖氨酸残基突变为精氨酸(Cse4K16R)创建了一个稳定的Cse4蛋白质(Collins等人，2004年)。Cse4K16R蛋白显着稳定，允许可溶性Cse4蛋白的积累。因为Cse4K16R仍然以着丝粒为靶点，所以它仍然与在着丝粒加载它的沉积因子相互作用。因此，我们构建了Cse4K16R-FLAG蛋白，并通过抗FLAG免疫沉淀纯化了它(数据未显示)。我们目前正在使用阴离子交换层析进行第二步纯化，并建议将纯化的蛋白质提交给质谱仪，以鉴定相互作用的蛋白质。我们随后将测试所鉴定的蛋白质是否与野生型Cse4蛋白相互作用，并帮助其在酵母着丝粒上装载。综上所述，这些研究应该确定导致着丝粒Cse4沉积的蛋白质。 Collins，K.A.，S.Furuyama和S.Biggins。2004年。蛋白质的降解有助于酵母Cse4/CENP-A组蛋白H3变体的唯一着丝粒定位。Curr Biol.14(21)1968-72年。