TOOLS FOR STUDYING EUKARYOTIC CELL MEMBRANE TRAFFICKING

研究真核细胞膜运输的工具

• 批准号: 2024240
• 负责人: Alexey G Zdanovsky
• 金额: $ 9.93万
• 依托单位: PROMEGA CORPORATION
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2024240
• 关键词: Clostridium; bacterial toxins; cell membrane; cell motility; gene expression; intracellular transport; method development; mutant; neurotoxins; site directed mutagenesis

DESCRIPTION (Adapted from applicant's abstract): Clostridial neurotoxins (NTs) have been extremely valuable for elucidating the mechanisms of neurotransmitter secretion from nerve cells since eight distinct NTs were found to act as specific proteases on only three neurosynaptic proteins: synaptobrevin, syntaxin and SNAP-25. However, a novel perspective has recently emerged from studies of NT action in non-neural cells. Once believed to be neural-specific, NTs introduced into the cytosol of non- neural cells by bypassing neural-specific cell-surface receptors are found to inhibit aspects of membrane trafficking. While surprising, these findings are understandable since a number of non-neural homologs of NT substrate proteins have been identified. The goals of this project are to develop a safe, efficient and general method for inducibly expressing Clostridial NT light chains in nonneural cells, and to employ this method to characterize the range of NT action on membrane trafficking in eukaryotic cells. The work proposed here will lead to the identification of specific membrane trafficking steps that are interrupted by NTs and to the characterization of NT substrates involved in these steps. Additional mutagenesis experiments are proposed to generate novel NT variants that have acquired a new substrate range. This work will also provide general tools for cell biological studies of commercial potential for investigating membrane trafficking in eukaryotic cells.

性状（改编自申请人摘要）：梭菌神经毒素 (NTs)对于阐明 从神经细胞的神经递质分泌，因为八个不同的NT是 发现仅在三种神经突触蛋白上起特异性蛋白酶的作用： 突触泡蛋白、突触融合蛋白和SNAP-25。 然而，一个新的视角， 最近从非神经细胞中NT作用的研究中出现。 一旦 被认为是神经特异性的，NT被引入非神经细胞的胞质溶胶中， 神经细胞通过绕过神经特异性细胞表面受体， 发现抑制膜运输的方面。 令人惊讶的是，这些 这些发现是可以理解的，因为NT的一些非神经同源物 底物蛋白已被鉴定。 该项目的目标是 开发一种安全、高效、通用的诱导表达方法 在非神经细胞中的梭菌NT轻链，并采用这种方法 为了表征NT对膜运输的作用范围， 真核细胞 这里提出的工作将导致识别 特定的膜运输步骤被NT中断， 这些步骤中涉及的NT底物的表征。 额外 提出了诱变实验以产生新的NT变体， 获得了一个新的底物范围。 这项工作还将提供一般 具有商业潜力的细胞生物学研究工具， 研究真核细胞的膜运输。