Nanoelectrochemical Study of Molecular Transport through the Nuclear Pore Complex

项目摘要 在这个方案中，我们结合了两种强大的纳米技术，即纳米体和纳米级扫描 电化学显微镜(SECM)，以获得前所未有的了解分子传输通过 核孔复合体(NPC)，是真核细胞中细胞质和细胞核之间的唯一通道。我们 从骆驼衍生的针对鼻咽癌不同成分的重链抗体中设计纳米抗体，即， 核孔素，创新性地检验我们的假设，这些假说在生物学和 实际应用于生物医学，以协同推进人类健康保健。拟议的工作开创了 抗核孔蛋白纳米体作鼻咽癌选择性阻滞剂的应用 核孔素在调节核质分子转运中的作用。我们应用纳米物体来测试我们的 最初的假设是核孔素通过NPC纳米孔不均匀地分布构成 中枢和外周通路。我们使用我们实验室开发的纳米级SECM来空间解析 纳米体-基于对小探针离子的低和高被动渗透率的阻断和畅通的途径， 从而在纳米孔内定位每个核孔素。此外，我们将纳米实体应用于 评估我们的新假设，即核孔素具有不同的疏水和带电氨基 酸将不同的大分子分类到不同的路径中，而不仅仅是通过疏水相互作用 这是一个长期的共识，但与静电相互作用是协同的。我们通过以下方式挑战这一共识 基于疏水脯氨酸和阳离子的神经毒性多肽的被动转运研究 精氨酸，最近被发现可以阻止鼻咽癌，作为遗传的潜在共同原因 神经退行性疾病。我们使用SECM来测定NPC对一种脯氨酸的高渗透性。 精氨酸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 受与核孔蛋白互补或竞争结合的纳米体的不同影响 多二肽。因此，这项研究将提供核孔蛋白的多肽靶向在 除了多肽-核孔蛋白相互作用的类型和强度。被动语态的SECM研究 输运为研究被动不透性大分子的荧光输运奠定了基础， 它可以通过NPC由核运输受体，即重要蛋白，作为关键的一步 基因表达调控和基因传递。我们使用纳米体来确定是否存在重要蛋白 利用疏水和阴离子结合的伴侣大分子通过外周途径 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，我们还评估 多肽的神经毒性是否与其阻断输入素促进的能力有关 大分子运输。总体而言，拟议的工作将为以下方面提供全新的化学见解 通过全国人大推进高效安全核进口基因治疗药物的合理设计。

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.