Nanoelectrochemical Study of Molecular Transport through the Nuclear Pore Complex

项目摘要 在这个提议中，我们将联合收割机两种强大的纳米技术结合起来，即，纳米抗体和纳米扫描 电化学显微镜（SECM），以获得前所未有的了解分子运输，通过 核孔复合体（NPC）是真核细胞胞质和核之间的唯一通道。我们 从骆驼衍生的抗NPC的不同组分的重链抗体工程化纳米抗体，即， 核孔蛋白，以创新的方式检验我们的假设，这在生物学上具有重要意义， 在生物医学领域协同推进人类健康。拟议的工作开创了 抗核孔蛋白纳米抗体作为NPC的选择性阻断剂的应用，以揭示每种纳米抗体的独特作用 核孔蛋白在调节核质分子转运中的作用。我们应用纳米抗体来测试我们的 核孔蛋白通过NPC纳米孔不均匀分布以构成 中枢和外周通路。我们采用我们实验室开发的纳米级SECM来空间分辨 基于对小探针离子的低和高被动渗透性的纳米抗体阻断和未阻断途径， 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，即核孔蛋白具有各种群体的疏水性和带电的氨基 酸将不同的大分子分类到不同的途径，而不仅仅是通过疏水相互作用 作为一个长期的共识，但与静电相互作用合作。我们挑战共识， 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，这是最近发现，以阻止NPC作为一个潜在的共同原因，遗传 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 受到与核孔蛋白互补或竞争结合的纳米抗体的不同影响。 聚二肽因此，本研究将提供由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之外。这些SECM研究的被动 传输为被动不可渗透大分子的荧光传输研究奠定了基础， 其可以通过核转运受体，即，importins，作为一个关键步骤， 基因表达调控和基因递送。我们使用纳米抗体来确定是否输入 通过利用疏水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 大分子运输总的来说，拟议的工作将提供从根本上新颖的化学见解， 推进基因治疗的合理设计，通过NPC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.