Traceless Staudinger Ligation for Studying Ubiquitin-Mediated Protein Degradation

描述（由申请人提供）：所有真核生物都有一个基本的细胞器，蛋白酶体，用于降解错误折叠和不需要的细胞蛋白。蛋白质通过多聚泛素标签的翻译后修饰而被靶向降解，其中异肽键将泛素或靶蛋白上的赖氨酸侧链连接到远端泛素的C末端。通过Lys 48连接的四泛素链是有效的降解信号，最近的研究表明，一些杂聚物也构成降解信号。然而，大多数连接性的影响还没有完全表征，现有的方法无法获得所有潜在的、不同的多聚泛素标签的有用数量。无痕施陶丁格连接是一种连接多肽的化学方法。Raines实验室最近设计了一种在生理pH值的水性缓冲液中运行该反应的程序。该方法将进一步优化折叠的功能蛋白质，并用于生成定义长度和连接性的泛素聚合物。分支聚合物也将产生;分支聚泛素链似乎抑制蛋白酶体，但这种作用的表征是不完整的。应使用表征良好的蛋白Sid作为降解靶标。Sid p应标记有多种四泛素均聚物和杂聚物。活性蛋白酶体应从酵母中分离，并用于纯化Sid p靶标的降解试验。最终，这将使得能够确定什么样的多聚泛素连接性构成降解信号的“代码”。此外，这种化学标记方法将使蛋白酶体降解的限制被探测。蛋白酶体能够选择性降解多蛋白复合物中的一种蛋白。非天然靶标的多聚泛素化将挑战蛋白酶体分离和降解两个极其紧密结合的伴侣中的一个靶标。蛋白质RNase 1应用作降解靶标，因为它与蛋白质RI（RNase Inhibitor）具有异常稳定的相互作用（81天半衰期）。公共卫生相关性：泛素介导的蛋白质降解途径的功能障碍与多种人类疾病相关，包括多种类型的癌症和许多神经系统疾病。因此，进一步了解这一途径中发生的功能（和故障）对于开发药物和寻找这些疾病的治疗方法至关重要。

DESCRIPTION (provided by applicant): All eukaryotes have an essential organelle, the proteasome, for degrading misfolded and unneeded cellular proteins. Proteins are targeted for degradation by the posttranslational modification of a polyubiquitin tag, in which isopeptide linkages connect a lysine side chain on ubiquitin or on the target protein to the C-terminus of the distal ubiquitin. Tetraubiquitin chains linked through Lys48 are potent degradation signals, and recent studies indicate that some heteropolymers also constitute a degradation signal. The effects of most connectivities are not fully characterized, however, and existing methodologies cannot access useful quantities of all potential, distinct polyubiquitin tags. Traceless Staudinger Ligation is a chemical method for concatenating polypeptides. The Raines lab recently designed a procedure for running this reaction in aqueous buffer at physiological pH. This methodology shall be further optimized for folded, functional proteins, and used to generate ubiquitin polymers of defined length and connectivity. Branched polymers shall also be generated; branched polyubiquitin chains appear to inhibit the proteasome, but characterization of this effect is incomplete. The well-characterized protein Sid shall be used as the degradation target. Sid p shall be tagged with a variety of tetraubiquitin homopolymers and heteropolymers. Active proteasomes shall be isolated from yeast, and used in degradation assays with the purified Sid p targets. Ultimately, this will enable the "code" for what polyubiquitin connectivities constitute a degradation signal to be determined. Furthermore, this chemical tagging method will enable the limits of proteasomal degradation to be probed. The proteasome is capable of selectively degrading one protein in a multi-protein complex. Polyubiquitination of an unnatural target will challenge the proteasome to separate and degrade one target of two extremely tightly-bound partners. The protein RNase 1 shall be used as the degradation target, for it has an unusually stable interaction (an 81-day half-life) with the protein RI (RNase Inhibitor). PUBLIC HEALTH RELEVANCE: The malfunction of the ubiquitin-mediated protein-degradation pathway is correlated with a variety of human diseases, including multiple types of cancer and many neurological disorders. The further- understanding of the functions (and malfunctions) that occur within this pathway is therefore crucial to the development of drugs and finding treatments for these diseases.