Department of chemistry, Université du Québec à Montréal, Canada.
Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois (CERMO-FC), Faculté des sciences, Université du Québec à Montréal, Canada.
FEBS J. 2021 Aug;288(16):4849-4868. doi: 10.1111/febs.15796. Epub 2021 Mar 20.
Protein ubiquitination has been historically associated with protein degradation, but recent studies have demonstrated other cellular functions associated with substrate ubiquitination. Among the RING-type ubiquitin E3 ligase enzymes present in the human genome, RNF167 is a transmembrane protein located in endosomes and lysosomes and is implicated in controlling the endolysosomal pathway. Substrates of RNF167 have been identified, but the ubiquitin-conjugating E2 enzymes involved in the mechanism remain unknown. In this study, we describe the interaction between RNF167 and conjugating E2 enzymes. By means of in vitro autoubiquitination and binding assays, we show that RNF167 functionally interacts with many conjugating E2s, while fluorescence microscopy illustrates that these interactions occur in endosomes and lysosomes. Kinetic analyses of the interaction between RNF167 and selected conjugating E2 enzymes reveal submicromolar dissociation constants. The computed model of interaction between the RING domain of RNF167 and conjugating enzymes gives us insights on how RNF167 could interact with conjugating E2 enzymes. Furthermore, the results reveal that in vitro polyubiquitination of the AMPA-type glutamate receptor subunit GluA2, one of the RNF167's known substrates, is possible by the conjugating E2 enzyme UBE2N only after GluA2 has been primed by ubiquitin subsequent to the action of an initiating conjugating E2 enzyme functionally binding RNF167. Pharmacological inhibition of UBE2N in cultured hippocampal neurons diminishes AMPA-induced GluA2 ubiquitination. This study characterizes interacting partners of RNF167 and constitutes an initial step toward the identification of functional pairs assembled from RNF167 and ubiquitin-conjugating E2 enzymes involved in the ubiquitination of RNF167's substrate.
蛋白质泛素化一直与蛋白质降解有关，但最近的研究表明，底物泛素化与其他细胞功能有关。在人类基因组中存在的 RING 型泛素 E3 连接酶中，RNF167 是一种位于内体和溶酶体中的跨膜蛋白，与控制内溶酶体途径有关。已经鉴定出 RNF167 的底物，但参与该机制的泛素结合 E2 酶尚不清楚。在这项研究中，我们描述了 RNF167 与连接酶之间的相互作用。通过体外自动泛素化和结合测定，我们表明 RNF167 与许多连接酶具有功能相互作用，而荧光显微镜则说明了这些相互作用发生在内体和溶酶体中。对 RNF167 与选定连接酶之间相互作用的动力学分析表明，解离常数为亚微摩尔。RNF167 的 RING 结构域与连接酶之间相互作用的计算模型使我们深入了解 RNF167 如何与连接酶相互作用。此外，结果表明，在体外，只有在 AMPA 型谷氨酸受体亚基 GluA2 被连接酶 UBE2N 多泛素化后，该亚基才能被 GluA2 泛素化，因为在起始连接酶之后，GlUA2 被连接酶 UBE2N 泛素化。这种起始连接酶能够与 RNF167 结合。在培养的海马神经元中，UBE2N 的药理学抑制可减少 AMPA 诱导的 GluA2 泛素化。这项研究描述了 RNF167 的相互作用伙伴，是鉴定与 RNF167 和参与 RNF167 底物泛素化的泛素结合 E2 酶组装的功能对的初步步骤。
