Traceless Staudinger Ligation for Studying Ubiquitin-Mediated Protein Degradation

描述(申请人提供)：所有真核生物都有一个重要的细胞器，即蛋白酶体，用于降解错误折叠和不需要的细胞蛋白质。蛋白质是通过多泛素标签的翻译后修饰而被降解的，在该标签中，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Lys48连接的四泛素链是强有力的降解信号，最近的研究表明，一些杂多聚体也构成了降解信号。然而，大多数连接性的影响还没有得到充分的表征，现有的方法无法获得所有潜在的、不同的多泛素标签的有用数量。无迹Staudinger配基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的水溶液中进行这一反应的程序。这一方法将进一步优化折叠的功能蛋白质，并用于产生确定长度和连接性的泛素聚合物。也应该产生支化聚合物；支化的多泛素链似乎抑制了蛋白酶体，但这种影响的表征是不完整的。应使用特性良好的蛋白质SID作为降解靶标。SID P应使用各种四泛素均聚物和杂聚体进行标记。活性蛋白酶体应从酵母中分离出来，并与纯化的SID P靶标一起用于降解分析。最终，这将使多泛素连接性构成降解信号的“代码”得以确定。此外，这种化学标记方法将使人们能够探索蛋白酶体降解的极限。蛋白酶体能够选择性地降解多蛋白质复合体中的一种蛋白质。非天然靶标的泛素化将挑战蛋白酶体分离并降解两个结合极为紧密的伙伴中的一个靶标。蛋白质RNase1应被用作降解目标，因为它与蛋白质RI(RNaseInhibitor)具有异常稳定的相互作用(半衰期为81天)。公共卫生相关性：泛素介导的蛋白质降解途径的故障与多种人类疾病有关，包括多种类型的癌症和许多神经疾病。因此，对这一途径中发生的功能(和故障)的进一步了解对于开发药物和寻找治疗这些疾病的方法至关重要。

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.