Madeline Zoltek, Angel L. Vázquez Maldonado, Xizi Zhang, Neville Dadina, Lauren Lesiak and Alanna Schepartz*,
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引用次数: 0
Abstract
The inefficient translocation of proteins across biological membranes limits their application as potential therapeutics and research tools. In many cases, the translocation of a protein involves two discrete steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules can achieve high protein uptake, but few are capable of efficient endosomal escape. One exception to this rule is ZF5.3, a mini-protein that exploits elements of the natural endosomal maturation machinery to translocate across endosomal membranes. Although some ZF5.3–protein conjugates are delivered efficiently to the cytosol or nucleus, overall delivery efficiency varies widely for different cargoes with no obvious design rules. Here we show that delivery efficiency depends on the ability of the cargo to unfold. Using fluorescence correlation spectroscopy, a single-molecule technique that precisely measures intracytosolic protein concentration, we show that regardless of size and pI, low-Tm cargoes of ZF5.3 (including intrinsically disordered domains) bias endosomal escape toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal, even if the Tm is high. These findings imply a novel pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo.
The cell-permeant mini-protein ZF5.3 crosses endosomal membranes most efficiently to deliver protein cargoes to the cell cytosol when the cargo is capable of unfolding under physiological conditions.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.