Short N-terminal disordered regions and the proline-rich domain are major regulators of phase transitions for full-length UBQLN1, UBQLN2 and UBQLN4.
Short N-terminal disordered regions and the proline-rich domain are major regulators of phase transitions for full-length UBQLN1, UBQLN2 and UBQLN4.
复制标题
短 N 端无序区域和富含脯氨酸的结构域是全长 UBQLN1、UBQLN2 和 UBQLN4 相变的主要调节因子。
DOI：
10.1101/2023.09.27.559790
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Castañeda,CarlosA
中科院分区：
文献类型：
--
作者：
Dao,ThuyP;Rajendran,Anitha;Galagedera,SarasiKK;Haws,William;Castañeda,CarlosA
高度同源的泛素结合穿梭蛋白 UBQLN1、UBQLN2 和 UBQLN4 在其特定的蛋白质质量控制功能以及定位于应激诱导的凝聚物、细胞聚集体和聚集体的倾向方面有所不同。我们之前表明 UBQLN2 在体外发生相分离，并且 UBQLN2 缺失构建体的相分离倾向与其在细胞中形成凝聚物的能力相关。在这里，我们证明全长 UBQLN1、UBQLN2 和 UBQLN4 在体外表现出不同的相行为。引人注目的是，UBQLN4 的相分离饱和浓度比 UBQLN1 低得多。然而，与 UBQLN2 不同，UBQLN1 和 UBQLN4 的相分离均不具有很强的温度依赖性。我们确定 UBQLN2 的温度依赖性相行为源于其独特的富含脯氨酸 (Pxx) 区域，而其他 UBQLN 中不存在该区域。我们发现 UBQLN1、UBQLN2 和 UBQLN4 的短 N 端无序区域通过静电相互作用抑制 UBQLN 相分离。 N 端区域的电荷变体表现出改变的相行为。与 UBQLN 相分离对 N 端区域组成的敏感性一致，放置在 UBQLN N 端的表位标签调节相分离。总体而言，我们的体外结果对细胞中 UBQLN 的研究具有重要意义，包括确定相分离作为区分 UBQLN 细胞作用的潜在机制，以及在使用表位标签时需要谨慎，以防止实验伪影。
Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts.