How ubiquitin-carrying enzymes contribute to ubiquitin ligase specificity

近年来，已经发明了新的药物，使致病蛋白和 一种名为泛素连接酶的酶，它能促进有问题的蛋白质的破坏。这种新药 医疗保健每年为行业提供10亿美元的资金，推动使用泛素连接酶治疗各种人类 疾病。这些努力中的大多数都是利用一种名为库林环连合酶(CRL)的酶家族。 CRL在人类中约有200名成员，共同控制着约20%的泛素依赖 蛋白质在细胞中的降解。因此，对这些酶是如何调节的认识是相当重要的。 吸引了广大观众的兴趣。已知人类CRL与至少7种额外的酶配对，这些酶 我们称之为泛素携带酶(UCES)，它是促进依赖CRL的蛋白质底物所必需的 退化。CRL的控制被认为主要是通过它们的可逆性决定的 用一种名为NEDD8的蛋白质进行修饰。此外，人们还认为，UCES的行为是混乱的。 对于CRL，这将排除CRL-UCE相互作用水平上的CRL调节。然而，CRL-UCE 活性CRL的最新结构强烈地暗示了特异性。我们的R01赠款的初步结果 表明CRL-UCE的特异性使这些配对具有异常快速的泛素转移速率， 潜在地提供了超越糖尿病的CRL功能的额外控制层。考虑到这些 观察，这项行政补充寻求获得一个停流仪器，以发展生物物理 蛋白质-蛋白质相互作用实验测定UCES间蛋白质复合体的形成动力学 和CRL。这些研究产生的结果将有助于在机械水平上解释CRL-UCE是如何 实现了专一性。

In recent years novel drugs have been invented that induce proximity between a disease-causing protein and an enzyme called a ubiquitin ligase that promotes the destruction of the problematic protein. This new drug modality is feeding a billion dollar per year industry push to employ ubiquitin ligases to treat various human diseases. Most of these efforts have been utilizing a family of enzymes called the Cullin-RING ligases (CRLs). With some 200 members in humans, the CRLs collectively control approximately 20 % of ubiquitin-dependent protein degradation in cells. As such, an appreciation for how these enzymes are regulated is of considerable interest to a very wide audience. Human CRLs are known to partner with at least 7 additional enzymes, which we refer to as ubiquitin-carrying enzymes (UCEs), that are required to promote CRL-dependent protein substrate degradation. The control of CRLs is believed to be determined predominantly through their reversible modification with a protein called NEDD8. Furthermore, it also was believed that UCEs act promiscuously towards CRLs which would preclude CRL regulation at the level of CRL-UCE interaction. However, CRL-UCE specificity is strongly implied by the recent structure of an active CRL. Preliminary results from our R01 grant indicate that CRL-UCE specificity endows these pairs with exceptionally rapid rates of ubiquitin transfer, potentially providing an additional layer of control of CRL function beyond neddylation. In consideration of these observations, this administrative supplement seeks to obtain a stopped-flow instrument to develop biophysical protein-protein interaction assays to measure the kinetics of formation of the protein complexes between UCEs and CRLs. The results generated from these studies will help explain on a mechanistic level how CRL-UCE specificity is achieved.