Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex.
Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex.
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DOI：
10.1371/journal.ppat.1008784
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发表时间：
2020-10
期刊：
影响因子：
6.7
通讯作者：
Mottram JC
中科院分区：
文献类型：
--
作者：
Burge RJ;Damianou A;Wilkinson AJ;Rodenko B;Mottram JC
翻译后修饰，如泛素化，对于协调利什曼原虫在区分其主要形态（promastigote和amastigote）时发生的细胞转化是重要的。在墨西哥利什曼原虫基因组中已鉴定出2个E1泛素激活（E1）、13个E2泛素结合（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，但目前对E1、E2和E3酶在该寄生虫中的作用知之甚少。利用CRISPR-Cas9对promastigote中产生的23个E1、E2和HECT/RBR E3零突变体进行Bar-seq分析，发现promastigote在无尾毛坯分化和哺乳动物感染过程中存在许多适应度缺失表型。E2s UBC1/CDC34、UBC2和UEV1以及HECT E3连接酶hec2是promastigote向amastigote成功转化所必需的，UBA1b、UBC9、UBC14、hec7和hec11是小鼠感染过程中正常增殖所必需的。在筛选中检测的所有泛素化酶无效突变体中，Δubc2和Δuev1在分化过程中表现出最极端的适应度损失。E1 UBA1a或E2s UBC3、UBC7、UBC12和UBC13不能产生零突变，这表明这些基因在proprostigotes中是必需的。人类UBE2N和UBE2V1/UBE2V2的同源物UBC2和UEV1的x射线晶体结构分析显示其异源二聚体具有高度保守的结构和界面。此外，重组L. mexicana UBA1a可以将泛素装载到UBC2上，使UBC2- uev1在体外形成k63连接的二泛素链。值得注意的是，UBC2可以在体外与人E3s RNF8和BIRC2协同形成非k63连锁的多泛素链，这表明UBC2可以独立于UEV1促进泛素化，但UBC2与UEV1结合会抑制这种能力。我们的研究证明了UBC2和UEV1在L. mexicana的分化和细胞内存活中的双重重要性，并表明这两个蛋白之间的相互作用对其泛素化活性和功能的调节至关重要。蛋白质的翻译后修饰是允许利什曼原虫在其昆虫媒介和哺乳动物宿主中存在的不同生命周期阶段之间转换的关键。特别是，泛素系统的组成部分对于利什曼原虫从昆虫（promastigote）到哺乳动物（amastigote）阶段的转化和小鼠的正常感染是重要的。然而，对利什曼原虫中产生泛素修饰的酶的作用知之甚少。在这里，我们描述了泛素化途径的28种酶，并表明许多酶是这种寄生虫的生命周期进展或小鼠感染所必需的。基于两个蛋白UBC2和UEV1在promastigoite向amastigoite转变中的重要性，我们选择了它们进行进一步的研究。我们证明了UBC2和UEV1形成了一个能够进行泛素化的异源二聚体，并且这种活性的结构基础在利什曼原虫、酿酒酵母和人类之间是保守的。我们还发现UBC2与UEV1的相互作用改变了UBC2泛素化活性的性质。总之，我们论证了泛素化酶在利什曼原虫生命周期和感染过程中的重要作用，并探讨了UBC2和UEV1功能的生物化学基础。
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. The post-translational modification of proteins is key for allowing Leishmania parasites to transition between the different life cycle stages that exist in its insect vector and mammalian host. In particular, components of the ubiquitin system are important for the transformation of Leishmania from its insect (promastigote) to mammalian (amastigote) stage and normal infection in mice. However, little is known about the role of the enzymes that generate ubiquitin modifications in Leishmania. Here we characterise 28 enzymes of the ubiquitination pathway and show that many are required for life cycle progression or mouse infection by this parasite. Two proteins, UBC2 and UEV1, were selected for further study based on their importance in the promastigote to amastigote transition. We demonstrate that UBC2 and UEV1 form a heterodimer capable of carrying out ubiquitination and that the structural basis for this activity is conserved between Leishmania, Saccharomyces cerevisiae and humans. We also show that the interaction of UBC2 with UEV1 alters the nature of the ubiquitination activity performed by UBC2. Overall, we demonstrate the important role that ubiquitination enzymes play in the life cycle and infection process of Leishmania and explore the biochemistry underlying UBC2 and UEV1 function.
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影响因子：
16
作者：
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通讯作者：
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影响因子：
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