Requirements for efficient endosomal escape by designed mini-proteins.
Requirements for efficient endosomal escape by designed mini-proteins.
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通过设计的微型蛋白质有效地逃逸内体的要求。
DOI：
10.1101/2024.04.05.588336
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Schepartz,Alanna
中科院分区：
文献类型：
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作者：
Giudice,Jonathan;Brauer,DanielD;Zoltek,Madeline;VázquezMaldonado,AngelL;Kelly,Mark;Schepartz,Alanna
ZF5.3 是一种结构紧凑、设计合理的微型蛋白，可有效地从多种细胞类型的内体中逃逸。尽管 ZF5.3 体积小（27 个氨基酸），但它可以从处理细胞的细胞质中完整分离出来，并引导多种类型的蛋白质进入细胞质和/或细胞核。在最好的情况下，递送效率达到或超过 50%，以建立 500 nM 或更高的核或胞质浓度。但除了需要可折叠的货物和完整的 HOPS 复合体之外，人们对 ZF5.3 如何穿过限制性内吞膜知之甚少。在这里，我们描述了 ZF5.3 能够实现有效内体逃逸的属性。我们确认 ZF5.3 在 pH 值 5.5 至 7.5 之间稳定，即使在高达 95 °C 的温度下也没有证据显示其发生折叠。此处还报道了 ZF5.3 在 pH 5.5 下的高分辨率 NMR 结构，显示了典型的 p 锌指折叠，其中五元基序无缝集成到 C 端 α 螺旋中。根据圆二色性和高分辨率 NMR 判断，在较低 pH 值下，ZF5.3 协同展开。解折叠发生在单个 Zn(II) 结合 His 侧链的质子化时，其 pKa 几乎与晚期内体腔的 pKa 完全一致。 pH 诱导的解折叠对于内体逃逸至关重要，因为尽管整体摄取较高，但在 pH 4.5 下保持折叠状态的 ZF5.3 类似物无法有效到达细胞质。最后，使用重构的脂质体，我们确定了 ZF5.3 与特定脂质 BMP 的高亲和力相互作用，该脂质选择性富集于晚期内体膜的内叶中。这种相互作用在低 pH 值下比中性 pH 值强 10 倍，这为解释为何优先发生逃逸并以 HOPS 依赖性方式从晚期内体区室中提供了分子图谱。这里确定的程序性内体逃逸的要求应该有助于和指导蛋白质、肽模拟物和其他大分子的设计，这些大分子能够完整地并以治疗相关的浓度到达胞质或核靶标。
ZF5.3 is a compact, rationally designed mini-protein that escapes efficiently from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.3 can be isolated intact from the cytosol of treated cells and guides multiple classes of proteins into the cytosol and/or nucleus. In the best cases, delivery efficiencies reach or exceed 50% to establish nuclear or cytosolic concentrations of 500 nM or higher. But other than the requirement for unfoldable cargo and an intact HOPS complex, there is little known about how ZF5.3 traverses the limiting endocytic membrane. Here we delineate the attributes of ZF5.3 that enable efficient endosomal escape. We confirm that ZF5.3 is stable at pH values between 5.5 and 7.5, with no evidence of unfolding even at temperatures as high as 95 °C. The high-resolution NMR structure of ZF5.3 at pH 5.5, also reported here, shows a canonical p zinc-finger fold with the penta-arg motif integrated seamlessly into the C-terminal α-helix. At lower pH, ZF5.3 unfolds cooperatively as judged by both circular dichroism and high-resolution NMR. Unfolding occurs upon protonation of a single Zn(II)-binding His side chain whose pKa corresponds almost exactly to that of the late endosomal lumen. pH-induced unfolding is essential for endosomal escape, as a ZF5.3 analog that remains folded at pH 4.5 fails to efficiently reach the cytosol, despite high overall uptake. Finally, using reconstituted liposomes, we identify a high-affinity interaction of ZF5.3 with a specific lipid–BMP–that is selectively enriched in the inner leaflet of late endosomal membranes. This interaction is 10-fold stronger at low pH than neutral pH, providing a molecular picture for why escape occurs preferentially and in a HOPS-dependent manner from late endosomal compartments. The requirements for programmed endosomal escape identified here should aid and inform the design of proteins, peptidomimetics, and other macromolecules that reach cytosolic or nuclear targets intact and at therapeutically relevant concentrations.