Wang Linna, Yang Li, Pan Li, Kadasala Naveen Reddy, Xue Liang, Schuster Robert J, Parker Laurie L, Wei Alexander, Tao W Andy
Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, United States.
Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, IN, 47907, United States.
J Am Chem Soc. 2015 Oct 14;137(40):12772-12775. doi: 10.1021/jacs.5b07875. Epub 2015 Oct 5.
Our understanding of the complex cell entry pathways would greatly benefit from a comprehensive characterization of key proteins involved in this dynamic process. Here we devise a novel proteomic strategy named TITAN (Tracing Internalization and TrAfficking of Nanomaterials) to reveal real-time protein-dendrimer interactions using a systems biology approach. Dendrimers functionalized with photoreactive cross-linkers were internalized by HeLa cells and irradiated at set time intervals, then isolated and subjected to quantitative proteomics. In total, 809 interacting proteins cross-linked with dendrimers were determined by TITAN in a detailed temporal manner during dendrimer internalization, traceable to at least two major endocytic mechanisms, clathrin-mediated and caveolar/raft-mediated endocytosis. The direct involvement of the two pathways was further established by the inhibitory effect of dynasore on dendrimer uptake and changes in temporal profiles of key proteins.
我们对复杂细胞进入途径的理解将极大地受益于对这一动态过程中关键蛋白的全面表征。在此，我们设计了一种名为TITAN（追踪纳米材料的内化和运输）的新型蛋白质组学策略，以利用系统生物学方法揭示实时蛋白质与树枝状大分子的相互作用。用光反应性交联剂功能化的树枝状大分子被HeLa细胞内化，并在设定的时间间隔进行照射，然后分离并进行定量蛋白质组学分析。通过TITAN总共确定了809种与树枝状大分子交联的相互作用蛋白，这些蛋白在树枝状大分子内化过程中以详细的时间方式被追踪到，至少可追溯到两种主要的内吞机制，即网格蛋白介导的内吞作用和小窝/脂筏介导的内吞作用。动力蛋白抑制剂对树枝状大分子摄取的抑制作用以及关键蛋白时间谱的变化进一步证实了这两条途径的直接参与。
