Regulation by post-translation modifications in response to stress

项目摘要/摘要 泛素连接酶(E3)代表了一组多样性和保守性的酶，有超过 600名会员。这些连接酶共同将小蛋白泛素连接到超过 25%的蛋白质组，从而调节每个蛋白质组的稳定性或活性 目标。尽管这套酶很重要，但只有一小部分 泛素连接酶具有广泛的生物学功能。我们已经进行了一次 CRISPR筛查人类编码基因突变的敏感性 泛素连接酶和脱泛素酶以一组抑制剂覆盖广泛 一系列生物途径。从这个屏幕上，我们鉴定了一种F盒蛋白，名为 FBXO42的突变使细胞对有丝分裂抑制物敏感，并导致 有丝分裂缺陷的细胞堆积。F盒蛋白是底物适配子 SCF-cullin环连接酶。泛素连接酶研究中的最大挑战之一 就是识别它们的底物。我们开发了几种方法来实现这一点，使用 (MS)光谱分析和荧光检测。Leah将鉴定有丝分裂底物 FBXO42采用“连接酶陷阱”融合蛋白，并通过遗传筛选。通过识别 FBXO42底物，我们将能够更好地了解它在细胞周期中的作用。

Project Summary/Abstract Ubiquitin ligases (E3s) represent a diverse and conserved group of enzymes, with over 600 members. These ligases collectively attach the small protein ubiquitin to more than twenty five percent of the proteome, thereby regulating the stability or activity of each target. Despite the importance of this set of enzymes, only a small percentage of ubiquitin ligases have well-characterized biological functions. We have conducted a CRISPR screen examining the sensitivity of mutations of genes encoding human ubiquitin ligases and deubiquitinating enzymes to a panel of inhibitors covering a broad range of biological pathways. From this screen, we identified an F box protein, called FBXO42, whose mutation renders cells sensitive to inhibitors of mitosis and causes the accumulation of cells with mitotic defects. F box proteins are substrate adaptors for the SCF cullin RING ligase. One of the greatest challenges in the study of ubiquitin ligases is identifying their substrates. We developed several methods to accomplish this, using (MS) spectrometry and fluorescent detection. Leah will identify mitotic substrates of FBXO42 using "ligase trap" fusion proteins and through genetic screens. By identifying FBXO42 substrates, we will be able to better understand its role in the cell cycle.