Role of protein unfolding in endosomal escape

蛋白质解折叠在内体逃逸中的作用

• 批准号: 2203903
• 负责人: Alanna Schepartz
• 金额: $ 56.7万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203903&HistoricalAwards=false
• 关键词: Role; protein; unfolding; endosomal; escape

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.

在化学系生命过程化学（CLP）项目的支持下，来自加州大学伯克利分校的Schepartz教授正在研究使某些蛋白质能够有效地穿过内体膜进入细胞溶质的过程。该过程通常被称为“内体逃逸”。内体逃逸知之甚少，代表了限制蛋白质治疗的细胞内效用的根本瓶颈。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。如果知道了这些设计规则，科学家们就可以开发和制备肽和蛋白质，并确信它们会到达预期的细胞内位置。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，因为它是直接定量内体逃逸而无需扩增的唯一方法。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采用。Schepartz团队提出了两个高度互补的活动（FCS BootCamp和五年级学生的BASIS课程），以扩大他们的科学影响力，超越现有的社区。两者都包括与创新、非正式科学交流、高等教育/专业发展和扩大参与有关的内容。大多数蛋白质和多肽绕过一条复杂的路径进入细胞质或细胞核。虽然一些环肽通过生物膜被动扩散，但这些分子是例外，而不是规则。对于大多数肽和蛋白质，进入胞质溶胶的过程涉及内吞作用，不仅需要内吞摄取，还需要内体逃逸。大多数被称为“细胞穿透肽”（CPP）的分子以10%的效率逃离内体。因此，尽管在肽模拟物和人工蛋白质的基础科学方面已经有了大量的杰出工作，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。被称为ZF5.3的多肽是一种28个氨基酸、无毒、从头设计的微型蛋白质，即使与蛋白质货物共价融合，也能以前所未有的效率（50%）运输到细胞质和细胞核。ZF5.3的显著活性需要五精氨酸基序-在β-螺旋上以限定阵列显示的五个Arg残基-和由HOPS（同型融合和蛋白分选）复合物（与内体转运相关的多聚体蛋白复合物）产生的独特门户。该提案将测试假设，即内体逃逸需要蛋白质和/或交付代理展开，并寻求确定生物物理标准，使蛋白质“交付”。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。