From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892.
From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892
J Biol Chem. 2018 Mar 16;293(11):3904-3912. doi: 10.1074/jbc.RA117.001322. Epub 2018 Feb 1.
March-I is a membrane-bound E3 ubiquitin ligase belonging to the membrane-associated RING-CH (March) family. March-I ubiquitinates and down-regulates the expression of major histocompatibility complex (MHC) class II and cluster of differentiation 86 (CD86) in antigen-presenting cells. March-I expression is regulated both transcriptionally and posttranslationally, and it has been reported that March-I is ubiquitinated and that this ubiquitination contributes to March-I turnover. However, the molecular mechanism regulating March-I ubiquitination and the importance of March-I's E3 ligase activity for March-I ubiquitination are not fully understood. Here we confirmed that, although March-I is ubiquitinated, it is not ubiquitinated on a lysine residue, as a lysine-less March-I variant was ubiquitinated similarly as wildtype March-I. We found that March-I E3 ligase activity is not required for its ubiquitination and does not regulate March-I protein expression, suggesting that March-I does not undergo autoubiquitination. Knocking down ubiquitin-conjugating enzyme E2 D1 (Ube2D1) impaired March-I ubiquitination, increased March-I expression, and enhanced March-I-dependent down-regulation of MHC-II proteins. Taken together, our results suggest that March-I undergoes lysine-independent ubiquitination by an as yet unidentified E3 ubiquitin ligase that, together with Ube2D1, regulates March-I expression.
March-I 是一种膜结合的 E3 泛素连接酶，属于膜相关环指（March）家族。March-I 泛素化并下调抗原呈递细胞中主要组织相容性复合体（MHC）II 类和分化群 86（CD86）的表达。March-I 的表达受到转录和翻译后调控的调节，据报道 March-I 被泛素化，这种泛素化有助于 March-I 的周转。然而，调节 March-I 泛素化的分子机制以及 March-I 的 E3 连接酶活性对 March-I 泛素化的重要性尚未完全了解。在这里，我们证实尽管 March-I 被泛素化，但它不是在赖氨酸残基上被泛素化，因为无赖氨酸的 March-I 变体与野生型 March-I 相似地被泛素化。我们发现 March-I 的 E3 连接酶活性不是其泛素化所必需的，也不调节 March-I 蛋白的表达，这表明 March-I 不发生自身泛素化。敲低泛素结合酶 E2 D1（Ube2D1）会损害 March-I 的泛素化，增加 March-I 的表达，并增强 March-I 依赖性 MHC-II 蛋白的下调。总之，我们的结果表明，March-I 经历了赖氨酸非依赖性泛素化，由尚未确定的 E3 泛素连接酶进行，该酶与 Ube2D1 一起调节 March-I 的表达。
