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

项目摘要(摘要) 一个被称为E3连接酶的广泛而多样的蛋白质家族识别将要成为 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，已有600多个E3连接酶 它们中的许多在底物识别模式上仍然是个谜。 药物发现中的一个新兴概念是使用小分子配体来招募 与E3连接酶相关的治疗相关蛋白质，导致泛素化和降解 被招募的蛋白质。与简单的抑制相比，这种策略提供了独特的优势。然而，一个主要的 靶向蛋白质降解的限制一直是E3可用于 底物的补充，以及最大尺寸的降解分子。因此，这些方面的进展 这些领域将显著扩大降解器设计的可能性，并提高 产生一种治疗有效的分子，同时降低有害的靶外效应的风险。 这项提议的长期目标是解密新的E3可以用来 被诱导通过小分子招募底物。这项应用的目的是获得分子 洞察E3连接酶DCAF16如何被小分子招募以及这如何影响其 细胞功能正常。中心假设是先前报道的化合物GNOE-0011 利用DCAF16作为E3连接酶来降解致癌蛋白BRD4，它可能被利用 将DCAF16招募到其他蛋白质靶点，包括难以药物治疗的转录因子。 为了解决这一假设，目标1将寻求从结构和生化特征 GNE-0011、DCAF16和BRD4之间的相互作用。原子级别的图景将为 底物补充，通过突变实验验证。这些交互作用将与 KB02，唯一已报道的DCAF16结合化合物。目标2将研究更广泛的生理学 研究较少的DCAF16的功能。虽然BRD4被Gne-0011降解，但有几种蛋白质是 被上调，可能代表了DCAF16的原始目标。调查这些目标， 结合无偏见的下拉实验，将提供对E3连接酶更好的理解 目标和活动，包括在疾病中可能发挥的作用。这两个目标加在一起将有助于我们的 对E3连接酶结构和功能的了解，极大地拓宽了E3连接酶设计的前景 新的靶向蛋白质降解剂分子。达纳-法伯癌症研究所的费舍尔实验室将 提供进行这项研究的最佳环境，这项工作将在细胞内提供培训 生物学、药物设计和蛋白质组学，这将极大地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.