Design, Synthesis, and Application of a Real-Time MAPK Activity Sensor

实时 MAPK 活动传感器的设计、合成和应用

• 批准号: 7680602
• 负责人: Cliff I Stains
• 金额: $ 4.72万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-11 至 2011-08-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680602
• 关键词: Acids; Amino Acids; Antibodies; Apoptosis; Be++ element; Beryllium; Cell Death; Cell division; Cell physiology; Cells; Communities; Confocal Microscopy; Consensus; Development; Disease; Docking; Evaluation; Fellowship; Fluorescence; Fluorescence Resonance Energy Transfer; Hydroxyl Radical; Length; Life; MAPK7 gene; Malignant Neoplasms; Medical; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Monitor; Nature; Pathway interactions; Peptide Synthesis; Peptides; Phase; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Polyethylene Glycols; Procedures; Proteins; Regulation; Reporting; Role; Signal Transduction; Site; Solid; Specificity; Staining method; Stains; Stimulus; Testing; Time; Variant; Western Blotting; Work; activating transcription factor; base; cell determination; chelation; controlled release; design; disease phenotype; extracellular; flexibility; fluorophore; genetic regulatory protein; inhibitor/antagonist; interest; kinase inhibitor; public health relevance; ratiometric; research study; response; sensor; small molecule; synthetic peptide

DESCRIPTION (provided by applicant): This proposal details the design, synthesis and application of a selective real-time Mitogen-Activated Protein Kinase (MAPK) activity probe. Phosphorylation plays a central role in the activation and deactivation of virtually all cellular processes. In particular, the MAPK signaling cascade produces a change in the observable phenotype of a cell by activating transcription factors in response to an extracellular stimulus. Recent work has shown that the use of CHelation-Enhanced Fluorescence can produce synthetic peptide-based phosphorylation sensors which undergo a 2- to 10-fold increase in fluorescence upon phosphorylation. These probes are modular in design and consist of an interchangeable kinase recognition motif, a phosphorylation site, and an unnatural amino acid. The modular nature of these probes allows for their extension to any kinase provided an appropriate recognition element is employed. Our specific aims are as follows: 1) Design, synthesis, and evaluation of a selective real-time MAPK activity sensor and 2) Delivery of the sensor into living cells. Thus this proposal will provide a fully modular synthetic set of kinase activity probes for use in real-time activity experiments in living cells. PUBLIC HEALTH RELEVANCE The Mitogen-Activated Protein Kinase (MAPK) pathway can stimulate a diverse set of cellular responses ranging from cell division to cell death; therefore even subtle changes in the regulation of this pathway can result in the acquisition of a disease state such as cancer. MAPKs are the terminal regulatory proteins in this pathway and as such are of great interest to the scientific and medical communities. The development of a selective real-time MAPK activity probe would aid in the evaluation of the duration and intensity of MAPK activity in response to external factors.

描述（由申请人提供）：本提案详细描述了选择性实时丝裂原活化蛋白激酶（MAPK）活性探针的设计、合成和应用。磷酸化在几乎所有细胞过程的激活和失活中起着核心作用。具体地，MAPK信号级联通过响应于细胞外刺激激活转录因子而产生细胞的可观察表型的变化。最近的工作表明，使用螯合增强荧光可以产生基于合成肽的磷酸化传感器，其在磷酸化后荧光增加2至10倍。这些探针在设计上是模块化的，并且由可互换的激酶识别基序、磷酸化位点和非天然氨基酸组成。这些探针的模块化性质允许它们延伸到任何激酶，只要采用适当的识别元件。我们的具体目标如下：1）设计、合成和评价选择性实时MAPK活性传感器和2）将传感器递送到活细胞中。因此，该提议将提供一套完全模块化的激酶活性探针，用于活细胞中的实时活性实验。促分裂原活化蛋白激酶（MAPK）通路可以刺激从细胞分裂到细胞死亡的多种细胞反应;因此，即使是该通路调节的微小变化也可能导致疾病状态的获得，例如癌症。MAPKs是该途径中的末端调节蛋白，因此引起科学和医学界的极大兴趣。选择性实时MAPK活性探针的开发将有助于评估响应于外部因素的MAPK活性的持续时间和强度。