Nanoelectrochemical Study of Molecular Transport through the Nuclear Pore Complex

项目概要 在这个提案中，我们结合了两种强大的纳米技术，即纳米抗体和纳米级扫描 电化学显微镜 (SECM)，通过 核孔复合体（NPC）是真核细胞细胞质和细胞核之间的唯一门户。我们 从骆驼衍生的重链抗体中设计出针对 NPC 不同成分的纳米抗体，即 核孔蛋白，创新地检验我们的假设，这些假设在生物学和 实际上在生物医学领域协同推进人类健康保健。拟议的工作开创了 应用抗核孔蛋白纳米抗体作为 NPC 的选择性阻断剂，揭示每种抗体的独特作用 核孔蛋白调节核细胞质分子运输。我们应用纳米抗体来测试我们的 最初的假设是核孔蛋白通过 NPC 纳米孔不均匀分布，构成 中枢和外周通路。我们采用实验室开发的纳米级 SECM 来空间解析 基于对小探针离子的低和高被动渗透性的纳米体阻断和未阻断途径， 分别，从而将每个核孔蛋白定位在纳米孔内。此外，我们将纳米抗体应用于 评估我们的新假设，即核孔蛋白具有不同的疏水性和带电氨基群体 酸不仅通过疏水相互作用将不同的大分子分类到不同的途径 作为长期共识，但与静电相互作用相配合。我们通过以下方式挑战共识 研究基于疏水性脯氨酸和阳离子的神经毒性聚二肽的被动转运 精氨酸，最近被发现可以阻断 NPC，这是遗传性疾病的潜在常见原因 神经退行性疾病。我们采用 SECM 来确定 NPC 对脯氨酸的高渗透性 – 精氨酸聚二肽及其具有各种疏水性或电荷的类似物。测得的渗透率将 与核孔蛋白互补或竞争性结合的纳米抗体对纳米抗体的影响不同 聚二肽。因此，本研究将提供聚二肽靶向的核孔蛋白的身份 除了聚二肽-核孔蛋白相互作用的类型和强度。这些 SECM 研究被动 运输为被动不可渗透大分子的荧光运输研究奠定了基础， 它可以通过核转运受体（即输入蛋白）陪伴通过 NPC，这是实现这一目标的关键一步。 基因表达调控和基因传递。我们使用纳米抗体来确定是否导入 利用疏水性和阴离子结合通过外周途径的伴侣大分子 识别具有疏水性和阳离子氨基酸的外周核孔蛋白的位点。此外，我们评估 聚二肽的神经毒性是否与其阻断输入促进的能力有关 大分子运输。总的来说，拟议的工作将为以下领域提供全新的化学见解： 通过NPC推进基因疗法的合理设计，以实现高效、安全的核输入。

Project Summary In this proposal, we combine two powerful nanotechnologies, i.e., nanobodies and nanoscale scanning electrochemical microscopy (SECM), to gain an unprecedented understanding of molecular transport through the nuclear pore complex (NPC) as the sole gate between the cytoplasm and nucleus of a eukaryotic cell. We engineer nanobodies from camelid-derived heavy-chain antibodies against distinct components of NPC, i.e., nucleoporins, to innovatively examine our hypotheses that are significant fundamentally in biology and practically in biomedicine to synergistically advance human health care. The proposed work pioneers the application of anti-nucleoporin nanobodies as selective blockers of NPC to reveal the unique role of each nucleoporin in the regulation of nucleocytoplasmic molecular transport. We apply nanobodies to test our original hypothesis that nucleoporins are heterogeneously distributed through the NPC nanopore to constitute central and peripheral pathways. We employ nanoscale SECM developed in our laboratory to spatially resolve nanobody-blocked and unblocked pathways based on low and high passive permeability to small probe ions, respectively, thereby locating each nucleoporin within the nanopore. Furthermore, we apply nanobodies to assess our new hypothesis that nucleoporins possess various populations of hydrophobic and charged amino acids to sort out different macromolecules into different pathways not exclusively by hydrophobic interactions as a long-standing consensus, but cooperatively with electrostatic interactions. We challenge the consensus by investigating the passive transport of neurotoxic polydipeptides based on hydrophobic proline and cationic arginine, which were recently found to block the NPC as a potential common cause of genetic neurodegenerative diseases. We employ SECM to determine the high permeability of NPC to a proline– arginine polydipeptide and its analogs with various hydrophobicity or charges. The measured permeability will be affected differently by nanobodies that bind nucleoporins complimentarily or competitively with polydipeptides. Accordingly, this study will provide the identity of nucleoporins targeted by polydipeptides in addition to the type and strength of polydipeptide–nucleoporin interactions. These SECM studies of passive transport lay the foundation for fluorescence transport studies of passively impermeable macromolecules, which can be chaperoned through the NPC by nuclear transport receptors, i.e., importins, as a crucial step to gene expression regulation and gene delivery. We employ nanobodies to determine whether importins chaperon macromolecules through the peripheral pathway by utilizing both hydrophobic and anionic binding sites to recognize peripheral nucleoporins with hydrophobic and cationic amino acids. In addition, we assess whether the neurotoxicity of polydipeptides is related to their capability to block importin-facilitated macromolecular transport. Overall, the proposed work will provide fundamentally novel chemical insights to advance the rational design of genetic therapeutics for efficient and safe nuclear import through the NPC.