A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.
A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins.
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DOI：
10.1371/journal.pbio.1001358
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发表时间：
2012
期刊：
影响因子：
9.8
通讯作者：
Steinfeldt T
中科院分区：
文献类型：
--
作者：
Fleckenstein MC;Reese ML;Könen-Waisman S;Boothroyd JC;Howard JC;Steinfeldt T
一种来自寄生性原生动物刚地弓形虫的分泌性激酶被证明与一种与免疫遗传学相关的假激酶合作，磷酸化和磷酸化IRG系统的小鼠抗性蛋白。小鼠抵抗原生动物寄生虫弓形虫感染的能力在很大程度上取决于一个复杂的非典型大GTP酶家族成员的功能，即干扰素-γ-诱导的免疫相关GTP酶（IRG蛋白）。然而，T.刚地弓形虫对小鼠是高毒力的，因为如最近所示，它们在细胞侵入时从棒状体分泌多态性蛋白激酶ROP 18进入宿主细胞胞质溶胶。根据等位基因，ROP 18可以作为T.通过磷酸化并由此使小鼠IRG蛋白失活而感染弓形虫。在这篇文章中，我们表明，IRG蛋白相互作用不仅与ROP 18，但也强烈与另一个多态性位点，ROP 5，已经牵连作为一个主要的毒力因子从遗传杂交的产品，但其功能以前一直是一个完整的谜。ROP 5蛋白是与ROP 18激酶相同的蛋白质家族的成员，但在序列、结构和功能上是假激酶。我们通过遗传和生物化学方法的组合表明，ROP 5蛋白作为ROP 18的重要辅助因子，并提供证据表明它们通过在IRG靶蛋白上强制执行非活性GDP依赖性构象而起作用。通过这样做，它们防止GTP依赖性活化，同时暴露开关I环上的靶苏氨酸用于通过ROP 18磷酸化，导致蛋白质的永久失活。这代表了一种新的机制，其中假激酶通过制备磷酸化底物而不是通过上调激酶本身的活性来促进伴侣激酶对靶标的磷酸化。 弓形虫是一种细胞内寄生的原生动物，感染了大约三分之一的人类。然而，人类不太可能是寄生虫进化的重要宿主，因为为了完成寄生虫的性周期，受感染的动物必须被猫吃掉。小鼠使用细胞内抵抗机制，IRG蛋白，对抗弓形虫。反过来，弓形虫似乎已经进化出一种毒力因子，一种称为ROP 18的蛋白激酶，它使IRG蛋白失活。我们发现ROP 18并不是单独起作用的。它需要ROP 5假激酶的帮助，ROP 5假激酶是与ROP 18相关但没有酶活性的蛋白质。ROP5假激酶通过结合IRG蛋白质来辅助ROP18，使其保持失活，并使其对酶攻击开放，并可能被ROP18激酶永久失活。这种机制说明了酶家族的成员可以失去其酶活性并进化为活性成员的调节剂或辅因子的原理。
A secreted kinase from the parasitic protozoan, Toxoplasma gondii, is shown to cooperate with a phylogenetically related pseudokinase to phosphorylate and inactivate a mouse resistance protein of the IRG system. The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself. Toxoplasma gondii is an intracellular parasitic protozoan infecting about a third of humankind. Humans, however, are unlikely to be important hosts in terms of the evolution of the parasite because, for completion of the parasite sexual cycle, the infected animal must be eaten by a cat. Therefore, important intermediate hosts for Toxoplasma are species like mice that are often preyed on by cats. Mice use an intracellular resistance mechanism, the IRG proteins, against Toxoplasma. In turn, Toxoplasma appears to have evolved a virulence factor, a protein kinase called ROP18, that inactivates IRG proteins. We show that ROP18 does not act alone. It needs help from the ROP5 pseudokinases, proteins related to ROP18 but without enzymatic activity. ROP5 pseudokinases assist ROP18 by binding to the IRG proteins, holding them inactive, and laying them open to enzymatic attack and likely permanent inactivation by the ROP18 kinase. This mechanism illustrates the principle that members of an enzyme family can lose their enzymatic activity and evolve into regulators or co-factors for the active members.
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影响因子：
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