Wada H, Kito K, Caskey L S, Yeh E T, Kamitani T
Division of Molecular Medicine, Department of Internal Medicine, and Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas- Houston Health Science Center, Houston,
Biochem Biophys Res Commun. 1998 Oct 29;251(3):688-92. doi: 10.1006/bbrc.1998.9532.
NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. To identify proteins that are involved in the NEDD8-conjugation and de-conjugation pathway, the yeast two-hybrid system was used to screen a human heart cDNA library using NEDD8 as a bait. Seven strongly positive clones were found to contain a cDNA insert encoding the ubiquitin C-terminal hydrolase, UCH-L3. In vitro GST pull-down assay demonstrated that UCH-L3 bound to both NEDD8 and ubiquitin. In contrast, UCH-L3 did not bind to sentrin-1, sentrin-2, or sentrin-3. Recombinant UCH-L3, but not UCH-L1, was able to cleave the C-terminus of NEDD8. Thus, UCH-L3 can function as a C-terminal hydrolase for both NEDD8 and ubiquitin. UCH-L3 may play a physiologically significant role in the cleavage of the C-terminus of NEDD8, which is required for NEDD8 to conjugate to target proteins.
NEDD8是一种新型泛素样蛋白，已被证明能以类似于泛素化和类泛素化的方式与核蛋白结合。为了鉴定参与NEDD8结合和去结合途径的蛋白质，利用酵母双杂交系统，以NEDD8为诱饵筛选人心脏cDNA文库。发现7个强阳性克隆含有编码泛素C末端水解酶UCH-L3的cDNA插入片段。体外GST沉降分析表明，UCH-L3与NEDD8和泛素均结合。相比之下，UCH-L3不与类泛素-1、类泛素-2或类泛素-3结合。重组UCH-L3而非UCH-L1能够切割NEDD8的C末端。因此，UCH-L3可作为NEDD8和泛素的C末端水解酶发挥作用。UCH-L3可能在NEDD8 C末端的切割中发挥生理上重要的作用，而这是NEDD8与靶蛋白结合所必需的。
