York Biomedical Research Institute and Department of Biology, University of York, United Kingdom.
Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, United Kingdom.
PLoS Pathog. 2020 Oct 27;16(10):e1008784. doi: 10.1371/journal.ppat.1008784. eCollection 2020 Oct.
Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
泛素化等翻译后修饰对于协调利什曼原虫寄生虫在其主要形态形式前鞭毛体和无鞭毛体之间分化时发生的细胞转化非常重要。在墨西哥利什曼原虫基因组中可以鉴定出2个E1泛素激活酶（E1）、13个E2泛素结合酶（E2）、79个E3泛素连接酶（E3）和20个去泛素化半胱氨酸肽酶（DUB）基因，但目前，关于E1、E2和E3酶在这种寄生虫中的作用知之甚少。使用CRISPR-Cas9对前鞭毛体中产生的23个E1、E2和HECT/RBR E3基因敲除突变体进行的Bar-seq分析揭示了在前鞭毛体向无鞭毛体分化和哺乳动物感染过程中许多适应性丧失的表型。E2s UBC1/CDC34、UBC2和UEV1以及HECT E3连接酶HECT2是从前鞭毛体成功转化为无鞭毛体所必需的，而UBA1b、UBC9、UBC14、HECT7和HECT11是小鼠感染期间正常增殖所必需的。在筛选中检测的所有泛素化酶基因敲除突变体中，Δubc2和Δuev1在分化过程中表现出最极端的适应性丧失。无法产生E1 UBA1a或E2s UBC3、UBC7、UBC12和UBC13的基因敲除突变体，这表明这些基因在前鞭毛体中是必需的。UBC2和UEV1分别是人类UBE2N和UBE2V1/UBE2V2的直系同源物，其X射线晶体结构分析揭示了一种具有高度保守结构和界面的异二聚体。此外，重组墨西哥利什曼原虫UBA1a可以将泛素加载到UBC2上，使UBC2-UEV1在体外形成K63连接的双泛素链。值得注意的是，UBC2可以在体外与人E3s RNF8和BIRC2合作形成非K63连接的多泛素链，表明UBC2可以独立于UEV1促进泛素化，但UBC2与UEV1的结合会抑制这种能力。我们的研究证明了UBC2和UEV1在墨西哥利什曼原虫的分化和细胞内存活中的双重必要性，并表明这两种蛋白质之间的相互作用对于调节它们的泛素化活性和功能至关重要。
