Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92307, United States.
Biochemistry. 2021 Mar 9;60(9):637-642. doi: 10.1021/acs.biochem.1c00067. Epub 2021 Feb 26.
DCAF16 is a substrate recognition component of Cullin-RING E3 ubiquitin ligases that can be targeted by electrophilic PROTACs (proteolysis targeting chimeras) to promote the nuclear-restricted degradation of proteins. The endogenous protein substates of DCAF16 remain unknown. In this study, we compared the protein content of DCAF16-wild type and DCAF16-knockout (KO) cells by untargeted mass spectrometry-based proteomics, identifying the Tudor domain-containing protein Spindlin-4 (SPIN4) as a protein with a level that was substantially increased in cells lacking DCAF16. Very few other proteomic changes were found in DCAF16-KO cells, pointing to a specific relationship between DCAF16 and SPIN4. Consistent with this hypothesis, we found that DCAF16 interacts with and ubiquitinates SPIN4, but not other related SPIN proteins, and identified a conserved lysine residue unique to SPIN4 that is involved in DCAF16 binding. Finally, we provide evidence that SPIN4 preferentially binds trimethylated histone H3K4 over other modified histone modifications. These results, taken together, indicate that DCAF16 and SPIN4 form a dedicated E3 ligase-substrate complex that regulates the turnover and presumed functions of SPIN4 in human cells.
DCAF16 是 Cullin-RING E3 泛素连接酶的底物识别组件，可被亲电子 PROTAC（蛋白水解靶向嵌合体）靶向，以促进蛋白质的核内限制性降解。DCAF16 的内源性蛋白质亚基仍然未知。在这项研究中，我们通过非靶向质谱蛋白质组学比较了 DCAF16 野生型和 DCAF16 敲除 (KO) 细胞的蛋白质含量，鉴定出含有 Tudor 结构域的蛋白质 Spindlin-4（SPIN4）是一种在缺乏 DCAF16 的细胞中水平显著增加的蛋白质。在 DCAF16-KO 细胞中发现的其他蛋白质变化很少，这表明 DCAF16 和 SPIN4 之间存在特定的关系。与这一假设一致，我们发现 DCAF16 与 SPIN4 相互作用并泛素化 SPIN4，但不与其他相关的 SPIN 蛋白相互作用，并鉴定出 SPIN4 中一个独特的保守赖氨酸残基参与 DCAF16 结合。最后，我们提供了证据表明 SPIN4 优先结合三甲基化组蛋白 H3K4，而不是其他修饰的组蛋白修饰。这些结果表明，DCAF16 和 SPIN4 形成了一种专门的 E3 连接酶-底物复合物，调节 SPIN4 在人类细胞中的周转率和假定功能。
