Muñoz-Braceras Sandra, Calvo Rosa, Escalante Ricardo
a Instituto de Investigaciones Biomedicas Alberto Sols; Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid ; Madrid , Spain.
Autophagy. 2015;11(6):918-27. doi: 10.1080/15548627.2015.1034413.
Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA(-), tipB(-), tipC(-), and tipD(-)). We found a clear autophagic dysfunction in tipC(-) and tipD(-) while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.
自噬缺陷在盘基网柄菌中会导致一种独特的表型，其特征是在丘状体阶段形成多个尖端。这促使我们分析先前描述的一些多尖端突变体（tipA(-)、tipB(-)、tipC(-)和tipD(-)）中的自噬情况。我们发现tipC(-)和tipD(-)存在明显的自噬功能障碍，而其他突变体则没有缺陷。tipD编码Atg16的同源物，这证实了该蛋白在盘基网柄菌自噬中的作用，并验证了我们的方法。tipC编码的蛋白与人类VPS13A（也称为舞蹈病蛋白）高度相似，其突变会导致舞蹈病-棘红细胞增多症综合征。尽管VPS13蛋白家族的成员在C末端存在与Atg2相似的区域，但此前没有成员与自噬相关。该区域还包含功能未知的保守结构域DUF1162。有趣的是，包含这两个基序的TipC C末端编码序列的表达在很大程度上弥补了突变体表型。缺乏TipC的盘基网柄菌细胞用标记物GFP-Atg18和GFP-Atg8观察到的自噬体数量减少，并且通过蛋白水解切割试验确定自噬降解受损。通过RNA干扰下调HeLa细胞中的人类VPS13A证实了该人类蛋白参与自噬。VPS13A缺失的细胞显示出自噬标记物的积累和自噬通量受损。
