Control of Macromolecular Traffic Through Plasmodesmata

通过胞间连丝控制大分子交通

• 批准号: 8662922
• 负责人: VITALY H CITOVSKY
• 金额: $ 2.52万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-04 至 2014-08-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8662922
• 关键词: Bacteria; Binding; Biological; Biological Models; Cells; Complex; Cytoplasm; Cytoplasmic Protein; Data; Down-Regulation; Elements; Enzymes; Eukaryota; Event; F-Box Proteins; Foundations; Genomics; HIV; Integration Host Factors; Invaded; Macromolecular Complexes; Mammalian Cell; Mammals; Mediating; Membrane; Modification; Molecular; Movement; Nanotubes; Nucleoproteins; Organelles; Outcome; Pathway interactions; Permeability; Phosphorylation; Plant Viruses; Plants; Plasmodesmata; Polysaccharides; Prions; Protein Kinase; Proteins; RNA; Regulation; Relaxation; Role; Sphincter; System; Testing; Tobacco Mosaic Virus; Ubiquitin; Vesicle; Viral; Virus; Virus Diseases; callose; intercellular communication; intercellular connection; macromolecule; multicatalytic endopeptidase complex; pathogen; protein activation; protein complex; protein transport; research study; tool; trafficking

DESCRIPTION (provided by applicant): Perhaps one of the most intriguing, yet least studied, aspects of intercellular transport in higher eukaryotes, from mammals to plants, is traffic of macromolecular complexes through intercellular cytoplasmic bridges between cells. These connections, termed tunneling nanotubes (TNTs) in mammals and plasmodesmata (Pd) in plants, are subverted by invading pathogens for their movement between the host cells. Whereas both mammalian and plant pathogens, e.g., prions and (potentially) HIV as well as most plant viruses, utilize cell-to-cell transport pathways, the first such capability was identified for plant viruses. Thus, viral transport via Pd represents a conceptual and mechanistic paradigm for intercellular traffic of macromolecules. We exploit Tobacco mosaic virus (TMV), whose Pd transport is mediated by its movement protein (MP), as a tool to study the regulatory mechanisms of Pd transport, focusing on two fundamental questions: (i) How does MP activate the host pathway for gating the Pd channel? And (ii) how does the host regulate this Pd-gating activity of MP? Our data suggest the presence of three regulatory mechanisms that involve MP: MP-induced activation of the cellular pathway for relaxation of a polysaccharide Pd sphincter, activation and deactivation of MP by phosphorylation, and down-regulation of MP by the host ubiquitin/proteasome system (UPS). These findings will be used to seek three objectives: Aim 1. Understand the mechanism by which MP gates Pd by modulating the polysaccharide sphincter. Our data identified a host cytoplasmic protein ANK that is recognized by MP and showed that the MP-ANK complexes accumulate at Pd and that the presence of ANK is required for MP-induced gating of Pd. We also showed that ANK interacts with ss-1, 3 glucanase (BG), an enzyme that degrades the polysaccharide Pd sphincter. We will test the hypothesis that MP redirects ANK from the cytoplasm to Pd, where ANK (or ANK-MP complexes) activates BG, that relaxes the Pd sphincter and elevates the Pd permeability. Aim 2. Understand the regulatory function of MP phosphorylation. We identified an ER- associated and Pd-associated protein kinases (ERPK and PdPK) that specifically phosphorylate MP, activating and deactivating its ability to gate Pd, respectively. We will explore the hypothesis that these PKs act as an "On/Off" switch of MP transport through Pd. Aim 3. Understand the role of the host UPS in down-regulation of MP. Our data show that challenge with pathogens induces expression of the plant defense-related F-box protein VBF. Among its pathogen-encoded substrates, VBF recognizes MP. We will test the hypothesis that VBF targets MP to proteasomal degradation via the SCFVBF pathway. Collectively, the expected outcomes of proposed experiments will define and characterize basic concepts and molecular mechanisms that underly activation and deactivation of intercellular transport of macromolecular complexes in general, and pathogens in particular.

描述（由申请人提供）： 从哺乳动物到植物，高等真核生物细胞间运输的最有趣但研究最少的方面之一可能是大分子复合物通过细胞间细胞质桥的运输。这些连接在哺乳动物中被称为隧道纳米管（TNT），在植物中被称为胞间连丝（Pd），它们在宿主细胞之间的运动被入侵的病原体破坏。而哺乳动物和植物病原体，例如，朊病毒和（潜在的）HIV以及大多数植物病毒利用细胞到细胞的转运途径，第一个这样的能力被鉴定为植物病毒。因此，通过Pd的病毒运输代表了大分子细胞间运输的概念和机制范例。我们利用烟草花叶病毒（Tobacco mosaic virus，TMV）的运动蛋白（Movement protein，MP）介导Pd的转运，研究了TMV中Pd转运的调控机制，主要研究两个基本问题：（1）MP如何激活宿主途径，从而门控Pd通道？以及（ii）宿主如何调节MP的Pd门控活性？我们的数据表明，涉及MP的三个监管机制的存在：MP诱导的激活的细胞通路的多糖Pd括约肌的松弛，激活和失活的MP磷酸化，和下调MP的主机泛素/蛋白酶体系统（UPS）。这些研究结果将用于寻求三个目标：目标1。了解MP通过调节多糖括约肌来控制Pd的机制。我们的数据确定了一个主机的细胞质蛋白ANK，这是由MP识别，并表明MP-ANK复合物积累在Pd和ANK的存在下，需要MP诱导的门控Pd。我们还表明，ANK与β-1，3葡聚糖酶（BG），一种降解多糖Pd括约肌的酶相互作用。我们将检验MP将ANK从细胞质重定向到Pd的假设，其中ANK（或ANK-MP复合物）激活BG，BG松弛Pd括约肌并提高Pd渗透性。目标二。了解MP磷酸化的调节功能。我们鉴定了特异性磷酸化MP的ER相关和Pd相关蛋白激酶（ERPK和PdPK），分别激活和失活其门控Pd的能力。我们将探讨的假设，这些PKs作为一个“开/关”开关MP运输通过Pd。目标3.了解主机UPS在MP下调中的作用。我们的数据表明，与病原体的挑战诱导植物防御相关的F盒蛋白VBF的表达。在其病原体编码的底物中，VBF识别MP。我们将检验VBF通过SCFVBF途径将MP靶向蛋白酶体降解的假设。总的来说，拟议的实验的预期成果将定义和表征基本概念和分子机制，基本的激活和失活的细胞间运输的大分子复合物一般，特别是病原体。