A DUAL E3 MECHANISM FOR RUB1 LIGATION TO CDC53

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Cullin RING连接酶（CRL）包括E3泛素连接酶的最大亚家族。 在人类中，六种cullin（CUL 1，2，3，4A，4 B和5），两种RBX家族RING蛋白（RBX 1和2）和数百种底物受体组装成不同的CRL，介导数千种靶点的泛素化，以调节大量的细胞过程。CRL的功能是由泛素样蛋白（UBL）NEDD 8连接到cullin的C-末端结构域中的保守赖氨酸来调节的。 NEDD 8既增强内在CRL泛素化活性，又阻止CRL与抑制剂CAND 1结合。 在人类中，已知NEDD 8级联包含单个E1（NAE 1-UBA 3）和两个E2（Ubc 12和UBE 2F）。 在酵母中，只有一个单一的E2，Ubc 12，已被发现与NEDD 8的直系同源物，Rub 1。 尽管NEDD 8/Rub 1激活CRL的重要性，但cullin连接NEDD 8/Rub 1的机制仍不完全清楚。 缺乏详细的机制观点，部分原因是两种不同的蛋白质被报道为NEDD 8/Rub 1连接Cul 1或其酵母直系同源物Cdc 53的E3。 一个候选E3是Rbx 1，其结合Cul 1/Cdc 53并具有RING结构域。 然而，Dcn 1也被鉴定为Rub 1 E3。Dcn 1晶体结构显示两个结构域，一个乌巴结构域和一个“增强neddylation”（PONY）结构域。据报道，单独的PONY结构域结合Ubc 12和Cdc 53，并且足以在体内和裂解物中增强Cdc 53-Rub 1水平。 基于这一发现，有人认为Rbx 1可能在Rub 1修饰cullin中发挥被动的结构作用。 然而，Cdc 53可以在没有Dcn 1的情况下在体外进行修饰，这就对Dcn 1作为E3的功能提出了疑问。 因此，我们正在剖析NEDD 8/Rub 1与Cul 1/Cdc 53连接的潜在机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cullin RING ligases (CRLs) comprise the largest subfamily of E3 ubiquitin ligases. In humans, six cullins (CUL1, 2, 3, 4A, 4B, and 5), two RBX-family RING proteins (RBX1 and 2), and hundreds of substrate receptors assemble into distinct CRLs that mediate ubiquitination of thousands of targets to regulate a vast array of cellular processes. CRL function is regulated by attachment of the ubiquitin-like protein (UBL) NEDD8 to a conserved Lys in a cullin's C-terminal domain. NEDD8 both enhances intrinsic CRL ubiquitination activity, and prevents CRL binding to the inhibitor CAND1. In humans, the NEDD8 cascade is known to contain a single E1 (NAE1-UBA3), and two E2s (Ubc12 and UBE2F). In yeast, only a single E2, Ubc12, has been found to work with the NEDD8 ortholog, Rub1. Despite the importance of CRL activation by NEDD8/Rub1, mechanisms underlying cullin ligation to NEDD8/Rub1 remain incompletely understood. A detailed mechanistic view is lacking, in part because two different proteins have been reported as being the E3 for NEDD8/Rub1 ligation to Cul1 or its yeast ortholog, Cdc53. One candidate E3 is Rbx1, which binds Cul1/Cdc53 and has a RING domain. However, Dcn1 was also identified as a Rub1 E3. The Dcn1 crystal structure revealed two domains, a UBA domain, and a "potentiating neddylation" (PONY) domain. The PONY domain alone was reported to bind Ubc12 and Cdc53, and is sufficient to enhance Cdc53~Rub1 levels in vivo and in lysates. Upon this discovery, it was suggested that Rbx1 may play a passive structural role in cullin modification by Rub1. However, Cdc53 can be modified in vitro without Dcn1, raising questions as to Dcn1's function as an E3. Thus, we are dissecting mechanisms underlying NEDD8/Rub1 ligation to Cul1/Cdc53.