Structural and functional investigation into protein degradation by the DCAF16 ubiquitin ligase

项目摘要（摘要） 一个广泛而多样的蛋白质家族称为E3连接酶，它识别要连接的靶蛋白。 泛素化，这标志着它的蛋白酶体降解。实际上，它们是决定 蛋白质的命运与蛋白质组的巨大多样性相一致，已经有超过600种E3连接酶被发现。 识别，其中大部分仍然是谜在其模式的基板识别。 药物发现中的一个新兴概念是使用小分子配体来招募一种新的药物。 将治疗相关蛋白质与E3连接酶连接，导致E3连接酶的泛素化和降解。 补充蛋白质。这种策略比简单的抑制提供了独特的优势。然而一个主要 靶向蛋白质降解的限制是可用于以下目的的E3的相对缺乏： 底物募集和大多数降解分子的大尺寸。因此，这些进展 区域将大大扩大降能器设计的可能性，并提高 产生治疗有效的分子，同时降低有害的脱靶效应的风险。 该提案的长期目标是解密新E3的结构原则， 诱导通过小分子募集底物。本申请的目的是获得分子量为10000的化合物。 深入了解E3连接酶DCAF 16如何被小分子招募以及如何影响其功能。 正常的细胞功能中心假设是，先前报道的化合物GNE-0011 利用DCAF 16作为E3连接酶来降解致癌蛋白BRD 4，并且它可以被利用 招募DCAF 16到其他蛋白质靶点，包括难以药物的转录因子。 为了解决这一假设，目标1将寻求从结构和生物化学上表征 GNE-0011、DCAF 16和BRD 4之间的相互作用。原子级的图像将为 底物募集，通过突变实验验证。这些相互作用将与 KB 02，唯一报道的DCAF 16结合化合物。目标2将研究更广泛的生理学 很少研究的DCAF 16的功能。虽然BRD 4被GNE-0011降解，但几种蛋白质被降解。 上调，可能代表DCAF 16的原始靶标。在调查这些目标时， 结合无偏的下拉实验，将提供一个更好的理解E3连接酶的 靶点和活性，包括在疾病中的可能作用。这两个目标将有助于我们 E3连接酶的结构和功能的知识，同时大大拓宽了设计的前景， 新的靶向蛋白质降解分子。Dana-Farber癌症研究所的Fischer实验室将 为开展这项研究提供最佳环境，这项工作将提供细胞培训 生物学、药物设计和蛋白质组学，这将大大增强和完善我现有的技能。

Project Summary (Abstract) A broad and diverse family of proteins called E3 ligases recognize the target protein that is to be ubiquitinated, which marks it for proteasomal degradation. In effect, they are a key factor in determining the fate of proteins. Consistent with the vast diversity of the proteome, over 600 E3 ligases have been identified, with much of them remaining enigmatic in their mode of substrate recognition. An emerging concept in drug discovery has been the use of small molecule ligands to recruit a therapeutically relevant protein to an E3 ligase, leading to the ubiquitination and degradation of the recruited protein. This strategy offers unique advantages over simple inhibition. However, a major limitation of targeted protein degradation has been the relative dearth of E3s that can be used for substrate recruitment, and the large size of most degradative molecules. Therefore, advances in these areas will significantly expand the possibilities of degrader design and improve the chances of generating a therapeutically effective molecule while reducing the risk of harmful off-target effects. The long-term goal of this proposal is to decrypt structural principles by which new E3s can be induced to recruit substrates via small molecules. The objective of this application is to gain molecular insights into how the E3 ligase DCAF16 can be recruited by small molecules and how that affects its normal cellular function. The central hypothesis is that the previously reported compound GNE-0011 utilizes DCAF16 as the E3 ligase to degrade the oncogenic protein BRD4, and that it may be utilized to recruit DCAF16 to other protein targets, including transcription factors that are difficult to drug. To address this hypothesis, Aim 1 will seek to structurally and biochemically characterize the interactions between GNE-0011, DCAF16, and BRD4. An atomic-level picture will provide a basis for substrate recruitment, validated by mutational experiments. These interactions will be compared with KB02, the only reported DCAF16-binding compound. Aim 2 will investigate the broader physiological function of the little-studied DCAF16. While BRD4 is degraded by GNE-0011, several proteins are upregulated, possibly representing the original targets of DCAF16. Investigating these targets, combined with unbiased pulldown experiments, will provide a better understanding of this E3 ligase’s targets and activity, including possible roles in disease. Together, these two aims will contribute to our knowledge of E3 ligase structure and function while greatly broadening the prospects for the design of new targeted protein degrader molecules. The Fischer Lab at the Dana-Farber Cancer Institute will provide an optimal environment to conduct this research and this work will provide training in cell biology, drug design, and proteomics that will greatly enhance and round out my existing skillset.