FRET-based Biosensors to Monitor Redox in Cell Cycle Regulation

基于 FRET 的生物传感器可监测细胞周期调节中的氧化还原

• 批准号: 8305728
• 负责人: Rex Gaskins
• 金额: $ 26.84万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305728
• 关键词: Anabolism; Apoptosis; Area; Biochemical; Biochemical Phenomena; Biological; Biological Models; Biosensor; Cancerous; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cell physiology; Cells; Cloning; Contact Inhibition; Coupling; Cultured Cells; DNA biosynthesis; Detection; Development; Dissection; Doxorubicin; Electron Transport; Energy Transfer; Engineering; Environment; Enzyme Activation; Equilibrium; Family suidae; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorouracil; Gases; Gene Expression; Generations; Genetic; Glutathione; Glutathione Disulfide; Glutathione Metabolism Pathway; HCT116 Cells; Homeostasis; Human; Imagery; Life; Link; Malignant Neoplasms; Measurement; Mediating; Methods; Modification; Molecular; Molecular Analysis; Monitor; Normal Cell; Organelles; Oxidation-Reduction; Performance; Pharmaceutical Preparations; Process; Protein Chemistry; Regulation; Relative (related person); Role; Signal Transduction; Staging; Sum; TP53 gene; Techniques; Technology; Time; Toxic effect; Tumor Cell Line; Visual; Work; base; cell growth; colon cancer cell line; cytotoxic; design; fluorophore; innovation; novel; oxidation; physical process; programs; public health relevance; ratiometric; response; sensor; tool; tumor

DESCRIPTION (provided by applicant): Cancer can be viewed as a state in which the balance between cell proliferation and cell death aberrantly favors the former. We and others have discovered that the intracellular redox environment exerts a profound influence on the normal cellular processes that regulate the balance between proliferation and cell death, including DNA synthesis, enzyme activation, cell cycle progression, proliferation, differentiation, and apoptosis. In fact, it could be argued that redox homeostasis is central to the governance of cell fate. Unfortunately, molecular mechanisms mediating redox sensitivity and regulation within cells are still poorly defined. Current pharmacological methods to alter intracellular redox state are limited by (i) their inability to operate independent of global biochemical alterations and cellular toxicity, and (ii) the required significant manipulation of culture conditions that perturb intracellular homeostasis. Our genetic constructs overcome these limitations as they enable real-time and extended assessment of alterations in intracellular redox without cellular disruption. These constructs use fluorescence resonance energy transfer (FRET), a distance- and orientation- dependent energy transfer process between donor and acceptor fluorophores. In these biosensors a change in redox induces a conformational change in the redox-sensitive switch that links the donor and acceptor, changing their distance, which in turn causes a detectable change in FRET efficiency. Here we propose to further define the sensitivity and dynamic range of our FRET biosensors relative to changes in the intracellular redox environment that appear to dictate cell fate. Advantages of this approach include: (1) the ability to quantify the change in redox state; (2) independence of sensor concentration; and (3) the ability to precisely tune the redox sensitivity and range by exchange of the switch or the fluorophore modules in the construct. Aim 1: Define the sensitivity and dynamic range of genetically engineered FRET redox biosensors during proliferation by comparison of nontransformed fibroblasts and isogenic porcine tumor cell lines with respect to the presence or absence of contact inhibition. Specifically, detection of physiologically relevant changes during successive stages of cell growth is proposed. Aim 2: Determine the extent to which the FRET biosensors are sensitive to changes in the intracellular redox environment of isogenic HCT116 p53+/+ and p53-/- cells treated with the chemotherapeutic drugs fluorouracil and doxorubicin in combination with perturbations in glutathione homeostasis. Specifically, the intracellular redox environment will be visualized in response to common chemotherapeutic drugs in combination with agents that modulate biosynthesis or metabolism of glutathione. Aim 3: Create second generation FRET biosensors that permit visual monitoring and dissection of intraorganellar local redox potentials. Specifically, we intend to quantify differences in redox potentials within subcellular organelles that are at a nonequilibrium steady-state with respect to each other in living cells. In sum, the proposed work will provide novel molecular tools that enable in depth examination of the role of redox signaling at the intracellular and intraorganellar level in cancer development. PUBLIC HEALTH RELEVANCE: This project pursues novel molecular tools-redox-sensitive biosensors-that will enable in depth examination of the role of redox signaling in cellular processes related to cancer development. Optimization of these biosensors will enable visualization of local changes in redox potential that might regulate progression through the cell cycle and mediate contact-dependent inhibition of cell growth, the disruption of which is a key hallmark of cancer. Ultimately, the tools will enhance understanding of the extent to which cancerous cells have lost the ability to mount changes in redox potential that accompany normal cell growth versus their sensitivity to these changes.

描述（由申请人提供）：可以将癌症视为一种状态，在这种状态下，细胞增殖和细胞死亡之间的平衡异常有利于前者。我们和其他人发现，细胞内氧化还原环境对调节增殖和细胞死亡之间平衡的正常细胞过程产生了深远的影响，包括DNA合成，酶激活，细胞周期进展，增殖，分化和凋亡。实际上，可以说氧化还原稳态对于细胞命运的治理至关重要。不幸的是，介导氧化还原敏感性和细胞内调节的分子机制仍然很差。当前改变细胞内氧化还原状态的药理方法受（i）无法独立于全球生物化学改变和细胞毒性独立于操作的限制，以及（ii）对扰动细胞内稳态的培养条件的重大操纵。我们的遗传构建体克服了这些局限性，因为它们能够实时和扩展对细胞内氧化还原改变而无需细胞破坏的变化。这些结构使用荧光共振能量传递（FRET），这是供体和受体荧光团之间的距离和方向依赖性能量转移过程。在这些生物传感器中，氧化还原的变化引起了氧化还原敏感开关的构象变化，该开关将供体和受体连接起来，改变其距离，进而导致FRET效率的可检测到变化。在这里，我们建议进一步定义FRET生物传感器的灵敏度和动态范围，相对于似乎决定细胞命运的细胞内氧化还原环境的变化。这种方法的优点包括：（1）量化氧化还原状态变化的能力； （2）传感器浓度的独立性； （3）通过交换开关或构造中的荧光团模块来精确调整氧化还原灵敏度和范围的能力。 AIM 1：通过比较非转化的成纤维细胞和等源性猪肿瘤细胞系在增殖过程中，定义基因工程的FRET氧化还原生物传感器的敏感性和动态范围，相对于存在或不存在接触抑制。具体而言，提出了在连续的细胞生长阶段的生理相关变化的检测。 目标2：确定fret生物传感器对等源性HCT116 p53+/+和p53 - / - 细胞用化学治疗药物氟尿嘧啶和黑霉素与粘膜固定稳态结合使用的化学治疗药物的细胞敏感的程度。具体而言，细胞内氧化还原环境将对常见的化学治疗药物进行可视化，并结合调节谷胱甘肽的生物合成或代谢的药物。 AIM 3：创建第二代FRET生物传感器，以允许对元素内局部氧化还原电位的视觉监测和解剖。具体而言，我们打算量化亚细胞细胞器内氧化还原电位的差异，这些细胞器中的氧化还原稳态相对于活细胞彼此之间的非平衡稳态。 总而言之，拟议的工作将提供新颖的分子工具，以深入研究氧化还原信号在细胞内和毛内层水平在癌症发展中的作用。 公共卫生相关性：该项目追求新颖的分子工具 - 雷斯敏感的生物传感器 - 可以深入研究氧化还原信号在与癌症发展相关的细胞过程中的作用。这些生物传感器的优化将使氧化还原电位的局部变化可视化，这可能通过细胞周期调节进展并介导接触依赖性细胞生长的抑制作用，其破坏是癌症的关键标志。最终，这些工具将增强对癌细胞在多大程度上失去正常细胞生长的氧化还原潜力变化能力与对这些变化的敏感性的能力的程度。

项目成果

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

区室化谷胱甘肽氧化还原电位对靶向 NQO1 的药理学醌的独特反应。

• DOI: 10.1016/j.bbrc.2016.12.082
• 发表时间: 2017
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Kolossov,VladimirL;Ponnuraj,Nagendraprabhu;Beaudoin,JessicaN;Leslie,MatthewT;Kenis,PaulJ;Gaskins,HRex
• 通讯作者: Gaskins,HRex

Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.

• DOI: 10.1258/ebm.2011.011009
• 发表时间: 2011-06-01
• 期刊: Experimental biology and medicine (Maywood, N.J.)
• 作者: Kolossov VL;Spring BQ;Clegg RM;Henry JJ;Sokolowski A;Kenis PJ;Gaskins HR
• 通讯作者: Gaskins HR

Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow.

• DOI: 10.1039/c0ib00071j
• 发表时间: 2011-03
• 期刊: Integrative biology : quantitative biosciences from nano to macro
• 作者: Lin C;Kolossov VL;Tsvid G;Trump L;Henry JJ;Henderson JL;Rund LA;Kenis PJ;Schook LB;Gaskins HR;Timp G
• 通讯作者: Timp G