Role of protein unfolding in endosomal escape

在化学系生命过程化学（CLP）项目的支持下，加州大学伯克利分校的Schepartz教授正在研究使某些蛋白质有效地穿过内体膜进入细胞质的过程。这个过程通常被称为“内体逃逸”。对内体逃逸了解甚少，是限制蛋白治疗在细胞内应用的基本瓶颈。Schepartz将测试内体逃逸需要蛋白质和/或递送剂展开的假设，并试图确定使蛋白质“可递送”的生物物理标准——设计规则。如果知道这些设计规则，科学家就可以开发和制备肽和蛋白质，并确信它们会到达预定的细胞内位置。被称为荧光相关光谱（FCS）的单分子技术是监测细胞内运输的最佳方法，因为它是唯一直接定量内体逃逸而无需扩增的方法。然而，设置是棘手的，数学是复杂的；两者都限制了它在学术界和工业界的应用。Schepartz团队提出了两个高度互补的活动（FCS BootCamp和针对五年级学生的BASIS课程），以将他们的科学影响扩展到现有社区之外。两者都包括与创新、非正式科学传播、高等教育/专业发展和扩大参与有关的要素。大多数蛋白质和多肽绕过一个复杂的路径进入细胞质或细胞核。虽然一些环肽被动地通过生物膜扩散，但这些分子是例外，而不是规则。对于大多数肽和蛋白质来说，到达细胞质的过程包括内吞作用，不仅需要内吞吸收，还需要内体逃逸。大多数被称为“细胞穿透肽”（CPPs）的分子以10%的效率逃离核内体。因此，尽管在模拟肽和人造蛋白质的基础科学方面有大量杰出的工作，但这些分子的效用主要局限于细胞表面。ZF5.3与大多数cpp不同。这种被称为ZF5.3的多肽是一种由28个氨基酸组成的、无毒的、全新设计的微型蛋白质，即使在与蛋白质货物共价融合的情况下，也能以前所未有的效率（50%）运输到细胞质和细胞核。ZF5.3的显著活性需要一个五精氨酸基序——五个精氨酸残基在一个限定的阵列上显示——和一个由HOPS（同型融合和蛋白质分选）复合物（一种与内体运输相关的多聚体蛋白质复合物）创建的独特入口。该提案将验证内体逃逸需要蛋白质和/或递送剂展开的假设，并试图确定使蛋白质“可递送”的生物物理标准。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professor Schepartz from the University of California, Berkeley is studying the process that enables certain proteins to traffic efficiently across endosomal membranes into the cytosol. This process is often referred to as “endosomal escape”. Endosomal escape is poorly understood and represents the fundamental bottleneck limiting the intracellular utility of protein therapeutics. Schepartz will test the hypothesis that endosomal escape demands protein and/or delivery agent unfolding and seeks to identify the biophysical criteria – the design rules – that make a protein “deliverable”. Were such design rules known, scientists could develop and prepare peptides and proteins with confidence that they would reach their intended intracellular location. The single molecule technique known as fluorescence correlation spectroscopy (FCS) is the very best way to monitor intracellular trafficking, as it is the only method that quantifies endosomal escape directly and without amplification. Yet the set-up is tricky and the math is complicated; both limit its adoption in academia and industry. The Schepartz team proposes two highly complementary activities (an FCS BootCamp and BASIS lesson for 5th graders) to extend the impact of their science beyond established communities. Both include elements related to Innovation, Informal Science Communication, Higher Education/Professional Development, and Broadening Participation. Most proteins and peptides circumnavigate a complex path to traffic into the cytosol or nucleus. Although some cyclic peptides diffuse passively through biological membranes, these molecules are the exception, not the rule. For most peptides and proteins, the journey to the cytosol involves endocytosis and demands not just endocytic uptake but also endosomal escape. Most molecules described as “cell-penetrating peptides” (CPPs) escape endosomes with efficiencies of 10%. As a result, although there has been an enormous amount of outstanding work on the basic science of peptidomimetics and artificial proteins, the utility of these molecules largely ends at the cell surface. ZF5.3 is unlike most CPPs. The polypeptide known as ZF5.3 is a 28-amino acid, non-toxic, de novo-designed mini-protein that traffics with unprecedented efficiency ( 50%) to the cytosol and nucleus, even when covalently fused to a protein cargo. The remarkable activity of ZF5.3 requires a penta-arginine motif–five Arg residues displayed in a defined array on an ⍺-helix–and a unique portal created by the HOPS (homotypic fusion and protein sorting) complex, a multimeric protein complex associated with endosomal transport. This proposal will test the hypothesis that endosomal escape demands protein and/or delivery agent unfolding and seeks to identify the biophysical criteria that make a protein “deliverable”.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.