Shakya Bikash, Kilili Geoffrey Kimiti, Wang Ling, Nakayasu Ernesto S, LaCount Douglas J
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
Microorganisms. 2022 Jul 16;10(7):1438. doi: 10.3390/microorganisms10071438.
proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein-protein interactions between erythrocyte cytoskeletal proteins and predicted exported proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with -deficient parasites.
蛋白质被输出到红细胞细胞质中，以创造一个支持寄生虫复制的环境。尽管预计有数百种蛋白质通过依赖和不依赖于输出元件（PEXEL）的机制输出，但输出蛋白质的功能在很大程度上尚未得到表征。在这项研究中，我们使用生化筛选方法来鉴定与从红细胞制备的内翻囊泡结合的假定输出蛋白质。在测试的69种含PEXEL基序的蛋白质中，有18种在两个或更多独立实验中与内翻囊泡（IOV）结合。通过共亲和纯化随后进行质谱分析、成对共纯化实验和分裂荧光素酶测定，我们鉴定了红细胞细胞骨架蛋白与预测的输出蛋白之间存在31种假定的蛋白质-蛋白质相互作用。我们进一步表明，PF3D7_1401600通过其MESA红细胞细胞骨架结合（MEC）基序与红细胞锚蛋白的血影蛋白结合结构域结合，并通过一个需要完整的螺旋穿插亚端粒（PHIST）结构域的片段与锚蛋白和4.1R的N末端结构域结合。将PF3D7_1401600引入红细胞血影中，在微滤过试验中增加了滞留率，这与先前报道的感染缺陷寄生虫的红细胞刚性降低的数据一致。
