Center for Infection and Immunity of Lille, Inserm U1019-CNRS UMR 8204, University of Lille Nord de France, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, 59019 Lille Cedex, France.
J Biol Chem. 2012 Jan 6;287(2):1306-21. doi: 10.1074/jbc.M111.276865. Epub 2011 Nov 28.
Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1.
越来越多的证据表明，调控蛋白磷酸酶 1（PP1）活性的蛋白质调节剂具有至关重要的功能，因为它们的缺失会严重影响细胞的生长和分裂。已经发现 PP1 在恶性疟原虫中是必不可少的，但对其调节剂知之甚少。在这项研究中，我们鉴定了 PP1 的抑制剂-3 的同源物，命名为 PfI3。NMR 分析表明 PfI3 属于无规卷曲蛋白家族。使用几种方法在体外证明了 PfI3 和 PfPP1 之间的高亲和力相互作用，表观解离常数 K(D)为 100nm。我们进一步表明，保守的（41）KVVRW（45）基序对于这种相互作用至关重要，因为将色氨酸（45）替换为丙氨酸（45）会严重降低与 PfPP1 的结合。令人惊讶的是，PfI3 无法挽救缺乏 I3（Ypi1）的酵母菌株。这种缺乏功能同源性的情况得到了支持，因为体外功能测定表明 PfI3 与酵母 I3 和人 I3 不同，它能增加 PfPP1 的活性。反向遗传学方法表明 PfI3 在血液阶段寄生虫的生长和/或存活中具有重要作用，因为试图获得敲除寄生虫的尝试均未成功，尽管 PfI3 的基因座是可接近的。GFP 标记的 PfI3 的主要定位于所有血液阶段寄生虫的核内，这与 PfI3 对核 PfPP1 活性的调节作用相一致。
