Facilitated Mechanisms in Zinc Regulation down to the Single-Molecule Level

单分子水平的锌调节促进机制

• 批准号: 9300948
• 负责人: Peng Chen
• 金额: $ 29.72万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9300948
• 关键词: Bacteria; Binding; Biochemical; Biological Sciences; Cell Survival; Cells; Chemicals; Collaborations; Communicable Diseases; Cues; DNA; DNA Binding; DNA-Protein Interaction; Development; Disease; Dissociation; Down-Regulation; Ensure; Escherichia coli; Excision; Foundations; Genetic Engineering; Genetic Transcription; Goals; Growth; Homeostasis; Human; Impairment; In Vitro; Invaded; Kinetics; Knowledge; Life; Measurement; Metals; Mission; Modeling; National Institute of General Medical Sciences; Organism; Outcome; Outcome Study; Pathway interactions; Pharmaceutical Preparations; Prevention; Proteins; Public Health; Regulation; Regulon; Repression; Research; Sulfhydryl Compounds; Testing; Therapeutic; Transcriptional Regulation; Weight; Zinc; Zinc deficiency; base; biophysical properties; chromatin immunoprecipitation; falls; fluorescence imaging; in vivo; innovation; insight; pathogen; physical science; public health relevance; response; single molecule; uptake

DESCRIPTION (provided by applicant): Zinc-sensing regulators enable cells to control Zn uptake, efflux, and storage that are critical for cell viability across all organisms. Many mechanistic pathways for these metalloregulators are still poorly understood. The long-term goal here is to understand how Zn homeostasis in the cell can be manipulated for preventative and therapeutic purposes. The overall objective here is to define how bacterial Zn-sensing metalloregulators (e.g., ZntR, Zur, and CzrA) ensure prompt switching between activation and deactivation and between repression and de-repression of their respective regulons in response to Zn excess or deficiency. The central hypothesis, formulated based on preliminary studies and past results, is that these metalloregulators use facilitated mechanisms for either protein dissociation from DNA or metal removal from the holo regulators in regulating transcription. To test this central hypothesis, the two PIs will collaborate and use the combined approach of single-molecule fluorescence imaging of protein-DNA interactions in vitro and in vivo, ensemble protein-DNA interaction kinetics, in vivo transcription profiling, chromatin immunoprecipitation, and protein and genetic engineering. The rationale for the proposed research is that the understanding of their regulation mechanisms will help in devising strategies and developing drugs to manipulate bacterial Zn uptake or efflux to limit their growth, thus contributing to the prevention and management of bacterial infectious diseases. The proposed research has three specific aims: (1) Identify the facilitated mechanisms of transcription deactivation by ZntR. The working hypotheses here are that the transcription deactivation of Zn efflux can occur via (#1) an assisted protein dissociation pathway, where free apo-ZntR, the repressor, assists the holo-ZntR, the activator, to dissociate from DNA, and/or (#2) a direct protein substitution pathway, where free apo-ZntR directly substitutes for the holo-ZntR on DNA. (2) Identify the facilitated mechanisms of transcription de-repression by Zur. The working hypotheses here are that (#3) free apo-Zur can assist holo-Zur, the repressor, to dissociate from DNA, leading to de-repression of Zn uptake, and (#4) bacillithiol (BSH) can facilitate Zn removal from DNA-bound holo-Zur to convert it to apo-Zur, which will dissociate from DNA leading to de-repression. (3) Identify the facilitated mechanisms of transcription de-repression and repression by CzrA. The working hypotheses here are that (#5) free holo-CzrA can assist apo-CzrA, the repressor, to dissociate from DNA, leading to de-repression of Zn efflux, and (#6) BSH can assist Zn removal from free holo-CzrA and accelerate subsequent CzrA binding to DNA for reestablishing repression. The research is significant because it will lead to the development of (bio)chemical strategies to manipulate bacterial Zn regulation to impair growth of pathogens and will fill knowledge gaps in Zn regulation mechanisms. The research is innovative because it integrates physical and biological sciences, combines both single-molecule and ensemble level measurements in vitro and in vivo, and introduces new concepts in transcription regulation.

描述（由申请人提供）：锌感应调节剂使细胞能够控制锌的吸收、流出和储存，这对于所有生物体的细胞活力至关重要。 Many mechanistic pathways for these metalloregulators are still poorly understood.这里的长期目标是了解如何操纵细胞中的锌稳态以达到预防和治疗目的。这里的总体目标是定义细菌锌感应金属调节剂（例如，ZntR、Zur 和 CzrA）如何确保响应锌过量或缺乏而在激活和失活以及抑制和去抑制之间迅速切换各自的调节子。根据初步研究和过去的结果制定的中心假设是，这些金属调节剂在调节转录时使用促进机制将蛋白质从 DNA 解离或从全息调节剂中去除金属。为了检验这一中心假设，两位 PI 将合作并使用体外和体内蛋白质-DNA 相互作用的单分子荧光成像、整体蛋白质-DNA 相互作用动力学、体内转录分析、染色质免疫沉淀以及蛋白质和基因工程的组合方法。这项研究的基本原理是，了解其调节机制将有助于制定策略和开发药物来操纵细菌锌的吸收或流出以限制其生长，从而有助于预防和管理细菌传染病。拟议的研究有三个具体目标：（1）确定 ZntR 促进转录失活的机制。这里的工作假设是，Zn 流出的转录失活可以通过 (#1) 辅助蛋白质解离途径发生，其中游离 apo-ZntR（阻遏物）协助 Holo-ZntR（激活剂）从 DNA 解离，和/或 (#2) 直接蛋白质替代途径，其中游离 apo-ZntR 直接替代 DNA 上的 holo-ZntR。 (2) 确定 Zur 促进的转录去抑制机制。这里的工作假设是，(#3) 游离的 apo-Zur 可以帮助阻遏物 Holo-Zur 从 DNA 上解离，从而导致锌吸收的去阻遏，而 (#4) 杆菌硫醇 (BSH) 可以促进 Zn 从 DNA 结合的 Holo-Zur 上去除，将其转化为 apo-Zur，从而与 DNA 解离，从而导致去阻遏。 (3) 确定CzrA 促进转录去抑制和抑制的机制。这里的工作假设是 (#5) 游离的 Holo-CzrA 可以帮助阻遏物 apo-CzrA 从 DNA 解离，导致 Zn 流出的去抑制，而 (#6) BSH 可以帮助 Zn 从游离的 Holo-CzrA 中去除，并加速随后的 CzrA 与 DNA 的结合，从而重新建立抑制。这项研究意义重大，因为它将导致（生物）化学策略的发展，以操纵细菌锌调节以损害病原体的生长，并将填补锌调节机制的知识空白。该研究具有创新性，因为它整合了物理和生物科学，结合了体外和体内的单分子和整体水平测量，并引入了转录调控的新概念。