Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom.
Mol Cell Proteomics. 2012 Mar;11(3):M111.014407. doi: 10.1074/mcp.M111.014407. Epub 2011 Oct 29.
The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin-proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8-cullin pathway.
NEDD8-Cullin E3 连接酶途径在蛋白质稳态中发挥着重要作用，特别是在细胞周期调节剂和转录控制网络的降解中。为了鉴定 NEDD8- 连接酶途径的靶蛋白，我们用小分子抑制剂 MLN4924 处理细胞后，进行了定量蛋白质组学分析。在多种人类细胞系中，NEDD8 抑制后，MRFAP1 及其相互作用蛋白 MORF4L1 是上调最明显的蛋白之一。我们发现，在没有 MLN4924 的情况下，MRFAP1 的周转率很快，并且通过泛素蛋白酶体系统降解。MLN4924 处理后 MRFAP1 丰度增加是由于降解率降低所致。对 MRFAP1 和 MORF4L1 结合蛋白的特征分析揭示了一个复杂的蛋白质-蛋白质相互作用网络。MRFAP1 与包括 CUL4B 在内的许多 E3 泛素连接酶结合，但不与包括 MRGBP 在内的 NuA4 复合物的成分结合，而 MRGBP 与 MORF4L1 结合。这些数据表明，MRFAP1 可能通过与 MORF4L1 结合，以与 MRGBP 相互排斥的方式调节 MORF4L1 与染色质修饰酶相互作用的能力。用特异性抗体对人组织进行 MRFAP1 免疫染色分析表明，它仅在少数组织中可检测到，特别是睾丸和大脑。引人注目的是，对睾丸生精小管的分析表明，在精原细胞中核染色最强，而在精母细胞和精子中染色较弱。MRGBP 与这些细胞类型中 MRFAP1 的表达呈负相关，这与细胞在睾丸减数分裂过程中相互作用的 MORF4L1 交换伙伴一致。这些数据突出了 NEDD8- 连接酶途径的一个重要新分支。
