Structure and Biochemistry of TRPA1:a noxious chemical and temperature sensor

TRPA1的结构和生物化学：有毒化学和温度传感器

• 批准号: 8651024
• 负责人: Monique Selina Jennifer Brewster
• 金额: $ 3.46万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8651024
• 关键词: Alternative Splicing; Analgesics; Ankyrin Repeat; Ankyrins; Anti Inflammatory Analgesics; Anti-Inflammatory Agents; Anti-inflammatory; Attention; Binding; Biochemical; Biochemistry; Biophysics; Calorimetry; Chemicals; Chronic; Circular Dichroism; Complement; Culicidae; Disease; Drug Design; Electrophysiology (science); Elements; Environment; Goals; Homology Modeling; Inflammation; Insecta; Investigation; Ion Channel; Knowledge; Link; Measures; Molecular; Mutagenesis; N-terminal; Neurons; Organism; Pain; Process; Property; Protein Isoforms; Proteins; Proteolysis; Research; Resolution; Role; Sensory; Structure; System; TRP channel; Targeted Research; Temperature; Temperature Sense; Testing; Well in self; Work; X-Ray Crystallography; base; design and construction; information gathering; insight; interest; mutant; painful neuropathy; public health relevance; receptor; research study; sensor; skills; three dimensional structure

DESCRIPTION (provided by applicant): The ability to detect and respond to harmful conditions in the environment is a skill essential for survival. As a result, many organisms have specialized systems for accurately detecting variables such as temperature or the presence of noxious chemicals. TRPA1, an ion channel in pain-sensing neurons, is involved in those processes. TRPA1 is activated by a wide range of electrophiles as well as by temperature. TRPA1 is a particularly interesting target for study due to its associations with pain and inflammation. The importance of the N-terminal cytoplasmic region of TRPA1 for temperature and chemical sensing is well established, but relatively little is known about the molecular mechanisms. This project focuses on the temperature-sensing mechanism of insect TRPA1. In insects, two TRPA1 isoforms differ only at a small region at the extreme N-termini, but the channels have very different temperature sensitivities12-13. The overall goal for this project is t understand how the N-termini of those insect TRPA1 isoforms interact with the ankyrin repeat domain to modulate temperature sensitivity. By combining biochemical, biophysical, and structural analyses of TRPA1 N-terminal fragments, I aim to elucidate the molecular mechanism of temperature sensing. The first aim of the project is investigate potential interactions between the isoform specific-regions of insect TRPA1 and the ankyrin repeat domain. This requires pure, soluble fragments of the N-terminal region of TRPA1, so l will put forth a large effort to produce the fragments necessary to search for interactions. Isothermal calorimetry and mutagenesis will be used to measure binding properties and identify regions important for interaction. In addition, pairs of corresponding fragments from the different insect TRPA1 isoforms will be produced for comparison in aim 2. The second aim is to identify biophysical and biochemical differences of the fragments that are responsible for the different temperature sensitivities of insect TRPA1 isoforms. Circular dichroism and limited proteolysis will be used to compare the secondary structure composition and thermal stability of construct pairs. The third and final aim is to solve the structures of TRPA1 N-terminal fragments through X-ray crystallography. By combining the information gained from aims 2 and 3, we aim to determine the role of the N-termini in the molecular mechanism of temperature sensing. Structures of the N-terminal domains of TRPA1 will provide a useful framework for further investigation of TRPA1 temperature sensing through biochemistry and electrophysiology experiments. Structural information will also be useful for rational drug design. TRPA1 antagonists may have analgesic and anti-inflammatory effects. These types of compounds may therefore be useful for treating neuropathic pain or chronic inflammation disorders.

描述（由申请人提供）：检测和应对环境中有害条件的能力是生存至关重要的技能。结果，许多生物具有专门的系统，可准确检测变量，例如温度或有害化学物质的存在。 TRPA1是疼痛感应神经元中的离子通道，参与了这些过程。 TRPA1被广泛的电力和温度激活。 TRPA1由于其与疼痛和炎症的关联，是研究的特别有趣的目标。 TRPA1的N末端细胞质区域在温度和化学传感方面的重要性已经很好地确定，但对分子机制的了解相对较少。该项目着重于昆虫TRPA1的温度感应机理。在昆虫中，两个TRPA1同工型仅在极端N末端的一个小区域有所不同，但是这些通道的温度敏感性非常不同。12-13。该项目的总体目标是了解那些昆虫TRPA1同工型的N末端如何与Ankyrin重复域相互作用以调节温度敏感性。通过结合TRPA1 N末端片段的生化，生物物理和结构分析，我旨在阐明温度传感的分子机制。 该项目的第一个目的是研究之间的潜在互动 昆虫TRPA1和Ankyrin重复域的同工型特异性区域。这需要TRPA1的N末端区域的纯净碎片，因此L将付出巨大的努力来产生寻找相互作用所需的片段。等温量热法和诱变将用于测量结合特性，并确定对相互作用重要的区域。此外，将生成来自不同昆虫TRPA1同工型的相应片段成对以在AIM 2中进行比较。第二个目的是鉴定碎片的生物物理和生化差异，这些片段的生物物理和生化差异负责昆虫TRPA1同工型的不同温度敏感性。圆二色性和有限的蛋白水解将用于比较构造对的二级结构组成和热稳定性。第三个也是最后一个目标是解决 通过X射线晶体学的TRPA1 N末端片段的结构。通过结合从目标2和3获得的信息，我们旨在确定N末端在温度传感的分子机理中的作用。 TRPA1的N末端结构域的结构将为通过生物化学和电生理实验进一步研究TRPA1温度传感的有用框架。结构信息也可用于理性药物设计。 TRPA1拮抗剂可能具有镇痛和抗炎作用。因此，这些类型的化合物可能对治疗神经性疼痛或慢性炎症疾病有用。