Veloria John R, Li Lin, Breen Gail A M, Goux Warren J
Department of Biological Sciences and ‡Department of Chemistry, The University of Texas at Dallas , Richardson, Texas 75080, United States.
ACS Chem Neurosci. 2017 Dec 20;8(12):2734-2745. doi: 10.1021/acschemneuro.7b00275. Epub 2017 Sep 6.
In the present study, a cell penetrating peptide (CPP)-amyloid conjugate was prepared (T-peptide), where the amyloid-forming sequence was homologous to a nucleating sequence from human Tau protein (VQIVYK). Kinetic and biophysical studies showed the peptide formed long-lived oligomers which were taken up by endocytosis and localized in perinuclear vesicles and in the cytoplasm of murine hippocampal neuroblastoma cells and human HeLa cells. Thioflavin S (ThS) staining of amyloid colocalized with pathological phosphorylated Tau, suggesting that the peptide was able to seed endogenous wild-type Tau. Subsequent experiments showed that aggregates present in the lysosomes mediated lysosome membrane permeability (LMP). We observed a decrease in total Tau, irrespective of phosphorylation state, consistent with Tau fragmentation by lysosomal proteases. We found cytotoxicity of T-peptide could be abrogated by inhibitors of lysosomal hydrolases and caspases, consistent with a model where Tau fragments processed by the lysosome leak into the cytoplasm and induce toxicity in subsequent downstream steps. It is our hope that the T-peptide system may prove amenable to the evaluation of small molecule inhibitors of cytotoxicity, especially those which target either Tau aggregation or the lysosomal/autophagy system.
在本研究中，制备了一种细胞穿透肽（CPP）-淀粉样蛋白缀合物（T-肽），其中形成淀粉样蛋白的序列与人 Tau 蛋白的成核序列（VQIVYK）同源。动力学和生物物理研究表明，该肽形成了长寿命的寡聚体，这些寡聚体通过内吞作用被摄取，并定位于小鼠海马神经母细胞瘤细胞和人 HeLa 细胞的核周囊泡和细胞质中。淀粉样蛋白的硫黄素 S（ThS）染色与病理性磷酸化 Tau 共定位，表明该肽能够引发内源性野生型 Tau 的聚集。随后的实验表明，存在于溶酶体中的聚集体介导了溶酶体膜通透性（LMP）。我们观察到总 Tau 减少，无论其磷酸化状态如何，这与溶酶体蛋白酶导致的 Tau 片段化一致。我们发现，溶酶体水解酶和半胱天冬酶的抑制剂可以消除 T-肽的细胞毒性，这与一种模型相符，即由溶酶体加工的 Tau 片段泄漏到细胞质中，并在随后的下游步骤中诱导毒性。我们希望 T-肽系统可能适用于评估细胞毒性小分子抑制剂，特别是那些靶向 Tau 聚集或溶酶体/自噬系统的抑制剂。
