Centre for Cardiovascular and Metabolic Research, The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK; Center for Biochemistry, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany.
Centre for Cardiovascular and Metabolic Research, The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK.
J Mol Biol. 2012 Oct 5;422(5):659-673. doi: 10.1016/j.jmb.2012.06.016. Epub 2012 Jun 15.
RhoBTB (BTB stands for broad-complex, tramtrack, bric à brac) proteins are tumor suppressors involved in the formation of cullin 3 (Cul3)-dependent ubiquitin ligase complexes. However, no substrates of RhoBTB-Cul3 ubiquitin ligase complexes have been identified. We identified MUF1 (LRRC41, leucine-rich repeat containing 41) as a potential interaction partner of RhoBTB3 in a two-hybrid screening on a mouse brain cDNA library. MUF1 is a largely uncharacterized protein containing a leucine-rich repeat and, interestingly, a BC-box that serves as a linker in multicomponent, cullin 5 (Cul5)-based ubiquitin ligases. We confirmed the interaction of MUF1 with all three mammalian RhoBTB proteins using immunoprecipitation. We characterized MUF1 in terms of expression profile and subcellular localization, the latter also with respect to RhoBTB proteins. We found out that MUF1 is a ubiquitously expressed nuclear protein that, upon coexpression with RhoBTB, partially retains in the cytoplasm, where both proteins colocalize. We also show that MUF1 is able to dimerize similarly to other leucine-rich repeat-containing proteins. To explore the significance of MUF1-RhoBTB interaction within Cul-ligase complexes and the mechanism of MUF1 degradation, we performed a protein stability assay and found that MUF1 is degraded in the proteasome in a Cul5-independent manner by RhoBTB3-Cul3 ubiquitin ligase complex. Finally, we explored a possible heterodimerization of Cul3 and Cul5 and indeed discovered that these two cullins are capable of forming heterodimers. Thus, we have identified MUF1 as the first substrate for RhoBTB-Cul3 ubiquitin ligase complexes. Identification of substrates of these complexes will result in better understanding of the tumor suppressor function of RhoBTB.
RhoBTB（BTB 代表 broad-complex、tramtrack、bric à brac）蛋白是参与形成 Cullin 3（Cul3）依赖性泛素连接酶复合物的肿瘤抑制因子。然而，尚未鉴定出 RhoBTB-Cul3 泛素连接酶复合物的底物。我们在小鼠脑 cDNA 文库的双杂交筛选中发现，MUF1（LRRC41，富含亮氨酸重复序列 41）是 RhoBTB3 的一个潜在相互作用伙伴。MUF1 是一种尚未充分表征的蛋白，含有亮氨酸丰富的重复序列，有趣的是，它还含有一个 BC 盒，作为多成分、基于 Cullin 5（Cul5）的泛素连接酶的连接子。我们使用免疫沉淀证实了 MUF1 与所有三种哺乳动物 RhoBTB 蛋白的相互作用。我们从表达谱和亚细胞定位方面对 MUF1 进行了表征，后者还涉及 RhoBTB 蛋白。我们发现 MUF1 是一种广泛表达的核蛋白，与 RhoBTB 共表达时，部分保留在细胞质中，两者在细胞质中发生共定位。我们还表明，MUF1 能够像其他富含亮氨酸重复序列的蛋白一样二聚化。为了探索 MUF1-RhoBTB 相互作用在 Cul 连接酶复合物中的意义以及 MUF1 降解的机制，我们进行了蛋白质稳定性测定，发现 MUF1 在蛋白酶体中以 Cul5 非依赖性方式被 RhoBTB3-Cul3 泛素连接酶复合物降解。最后，我们探索了 Cul3 和 Cul5 之间可能的异二聚化，并且确实发现这两个 Cullin 能够形成异二聚体。因此，我们鉴定出 MUF1 是 RhoBTB-Cul3 泛素连接酶复合物的第一个底物。鉴定这些复合物的底物将有助于更好地理解 RhoBTB 的肿瘤抑制功能。
