Schoijet Alejandra C, Sternlieb Tamara, Alonso Guillermo D
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres", Vuelta de Obligado 2490, Buenos Aires, C1428ADN, Argentina.
Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Intendente Güiraldes 2160, C1428EGA CABA, Argentina.
J Eukaryot Microbiol. 2017 May;64(3):308-321. doi: 10.1111/jeu.12367. Epub 2016 Sep 23.
Autophagy is a degradative process by which eukaryotic cells digest their own components to provide aminoacids that may function as energy source under nutritional stress conditions. There is experimental evidence for autophagy in parasitic protists belonging to the family Trypanosomatidae. However, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzi differentiation and thus might have a role in pathogenicity. Here, we report the cloning and biochemical characterization of TcVps15. In addition, we demonstrate that TcVps15 interact with the PI3K TcVps34 and that both proteins associate with cellular membranes. Under nutritional stress conditions, TcVps15 and TcVps34 modify their subcellular distribution showing a partial co-localization in autophagosomes with TcAtg8.1 and using an active site TcVps15-mutated version (TcVps15-K219D-HA) we demonstrated that this relocalization depends on the TcVps15 catalytic activity. Overexpression of TcVps15-HA and TcVps15-K219D-HA also leads to increased accumulation of monodansylcadaverine (MDC) in autophagic vacuoles under nutritional stress conditions compared to wild-type cells. In addition, the MDC-specific activity shows to be significantly higher in TcVps15-HA overexpressing cells when compared with TcVps15-K219D-HA. Our results reveal for the first time a role of TcVps15 as a key regulator of TcVps34 enzymatic activity and implicate the TcVps15-Vps34 complex in autophagy in T. cruzi, exposing a new key pathway to explore novel chemotherapeutic targets.
自噬是一种降解过程，通过该过程真核细胞消化自身成分以提供氨基酸，这些氨基酸在营养应激条件下可作为能量来源。有实验证据表明，属于锥虫科的寄生原生生物中存在自噬现象。然而，迄今为止，在这些寄生虫中，很少有参与这一过程的蛋白质得到表征。此外，已有研究表明自噬参与克氏锥虫的分化，因此可能在致病性方面发挥作用。在此，我们报告了克氏锥虫Vps15（TcVps15）的克隆及生化特性。此外，我们证明TcVps15与PI3K TcVps34相互作用，且这两种蛋白质均与细胞膜相关。在营养应激条件下，TcVps15和TcVps34改变其亚细胞分布，在自噬体中与TcAtg8.1呈现部分共定位，并且通过使用活性位点突变的TcVps15版本（TcVps15-K219D-HA），我们证明这种重新定位依赖于TcVps15的催化活性。与野生型细胞相比，在营养应激条件下，TcVps15-HA和TcVps15-K219D-HA的过表达也导致自噬泡中单丹磺酰尸胺（MDC）积累增加。此外，与TcVps15-K219D-HA相比，在过表达TcVps15-HA的细胞中，MDC特异性活性显著更高。我们的结果首次揭示了TcVps15作为TcVps34酶活性关键调节因子的作用，并表明TcVps15-Vps34复合物参与克氏锥虫的自噬过程，揭示了一条探索新型化疗靶点的新关键途径。
