Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana

IF 1.4 4区 医学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Rebecca J. Burge , Katie H. Jameson , Vincent Geoghegan , Adam A. Dowle , Jeremy C. Mottram , Anthony J. Wilkinson
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引用次数: 0

Abstract

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.

与墨西哥利什曼病的 UBC2 和 UEV1 形成功能性 E3 连接酶复合物。
在真核细胞中,分子命运和细胞反应是由多组分酶系统决定的,该系统将泛素和泛素样修饰物可逆地连接到目标蛋白质上。通过缺失诱变和生命周期表型研究,最近确定了墨西哥利什曼原虫中泛素蛋白酶体系统的范围及其对寄生虫生存的重要性。研究表明,泛素结合 E2 酶 UBC2 以及与之在体外形成稳定复合物的 E2 酶变体 UEV1 是原原体寄生虫分化为传染性非原体寄生虫所必需的。为了进一步研究,我们使用免疫沉淀 Myc-UBC2 或 Myc-UEV1 来鉴定 L. mexicana 原原体中的相互作用蛋白。UBC2 的相互作用组包括多种泛素-蛋白酶体成分,包括 UEV1 和四种 RING E3 连接酶,以及预测在碳水化合物代谢和细胞内运输中发挥作用的潜在底物。较小的 UEV1 相互作用组包括六个蛋白质,其中包括 UBC2 和 UBC2 相互作用组的共享成分,这与细胞内 UBC2-UEV1 复合物的存在是一致的。研究表明,重组的 RING1、RING2 和 RING4 E3 连接酶支持涉及 E1、UBA1a 和 UBC2 到可用底物蛋白或未锚定泛素链的泛素转移反应。这些研究确定了 UBC2 依赖性泛素化途径的其他组成部分,该途径以前曾被证明是原原体向非原体分化所必需的。
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来源期刊
CiteScore
2.90
自引率
0.00%
发文量
51
审稿时长
63 days
期刊介绍: The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths and their interactions with both the definitive and intermediate host. The main subject areas covered are: • the structure, biosynthesis, degradation, properties and function of DNA, RNA, proteins, lipids, carbohydrates and small molecular-weight substances • intermediary metabolism and bioenergetics • drug target characterization and the mode of action of antiparasitic drugs • molecular and biochemical aspects of membrane structure and function • host-parasite relationships that focus on the parasite, particularly as related to specific parasite molecules. • analysis of genes and genome structure, function and expression • analysis of variation in parasite populations relevant to genetic exchange, pathogenesis, drug and vaccine target characterization, and drug resistance. • parasite protein trafficking, organelle biogenesis, and cellular structure especially with reference to the roles of specific molecules • parasite programmed cell death, development, and cell division at the molecular level.
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