Near-infrared light activated protein photoswitches

近红外光激活蛋白质光开关

• 批准号: 8471674
• 负责人: Mark Gomelsky
• 金额: $ 14.23万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471674
• 关键词: Address; Adenylate Cyclase; Adhesions; Affect; Animal Model; Animals; Apoptosis; Biological Process; Blood Glucose; Cardiac; Cardiovascular Physiology; Caspase; Cell Differentiation process; Cell Survival; Cell physiology; Cells; Cellular biology; Chemicals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; Cysteine Protease; Data; Development; Developmental Biology; Diabetes Mellitus; Disease; Drosophila genus; Drosophila melanogaster; Engineering; Enzymes; Escherichia coli; Evaluation; Generations; Genes; Genetic; Genetic Screening; Government; Growth; Guanylate Cyclase; Homodimerization; Image; Immunology; In Vitro; Knowledge; Learning; Life; Light; Memory; Mission; Modeling; Mus; Neurobiology; Oncogenes; Output; Penetration; Persons; Pharmacologic Substance; Phosphotransferases; Photoreceptors; Phytochrome; Procedures; Process; Property; Proteins; Public Health; Recombinants; Research; Research Personnel; Resolution; Rhodobacter sphaeroides; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Source; Specificity; Stimulus; Study models; Testing; Tetrapyrroles; Time; Tissues; Transplantation; Variant; Visible Radiation; animal tissue; base; cancer cell; cell growth; cell killing; design; follow-up; gene therapy; in vivo; inhibitor/antagonist; innovation; insight; interdisciplinary approach; killings; model development; monomer; nervous system disorder; optogenetics; photoactivation; pro-caspase-3; protein function; research study; second messenger; small molecule; spatiotemporal; tool; tumor

DESCRIPTION (provided by applicant): The following contains proprietary/privileged information that M. Gomelsky requests not to be released to persons outside the Government, except for purposes of review and evaluation. PROJECT SUMMARY: Small molecule activators and inhibitors of signal transduction pathways are biologically useful, but are limited by their target specificities and spatiotemporal resolutio in vivo. A recently emerged optogenetic strategy can supplement chemical/pharmacological approaches. Ontogenetic involves introduction into cells and animals of genes encoding proteins whose activities can be photoactivated. Light is a unique stimulus in that it can control protein activities in vivo in a reversible manner and with spatiotemporal precision unattainable by chemicals. Photoreceptors of the bacteriophytochrome type absorb near-infrared light, which has superior tissue penetration properties, thus allowing protein photoactivation from unobtrusive external light sources. This is particularly important for studies on whole animals, such as mice. Recently, progress has been made in "transplanting" natural photoreceptor modules to control heterologous protein activities. Our long-term objective is to elucidate principles of engineering near-infrared light activated proteins using photosensory modules of bacteriophytochromes. This exploratory proposal will test the hypothesis that bacteriophytochrome photoreceptor domains can activate diverse homodimeric output activities. We will exploit our earlier studies of the BphG protein, a unique, near-infrared light activated diguanylyl cyclase. The nucleotidyl cyclases will be used as engineering targets. And an executioner (effectors) caspase rationally design bacteriophytochrome-based proteins, we will employ a multiprong approach involving circumventing our present inability to computational and structural analyses of proteins with genetic screening in E. coli. The proposed concept of engineering near-infrared light activated homodimeric proteins and the multidisciplinary approach to bacteriophytochrome engineering are innovative and feasible, as we already have constructed the first photoactivated homodimeric enzyme with a heterologous activity . Upon completion of this project, we anticipate to advance our understanding of the mechanism of light-induced signal propagation in bacteriophytochromes, and to uncover engineering principles for constructing homodimeric near-infrared light activated proteins. Because a large number of signaling proteins function as homodimers, light-induced protein homodimerization can be used to control a variety of cellular functions including apoptosis, differentiation, proliferation, transformation and adhesion. This research is significant because cAMP and cGMP control many cellular processes including growth, blood glucose levels, cardiac contractile function, and learning, memory and cancer cell survival. Photoactivated executioner caspase generated here will allow researchers to conduct targeted cell/tissue killing in whole animals using a mild and noninvasive procedure. These tools will likely find applications in cell biology, immunology and developmental biology, and potentially in cancer gene therapy.

描述（由申请人提供）：以下内容包含M. Gomelsky要求不将其释放给政府以外的人，除非是为了审查和评估的目的。 项目概要：信号转导途径的小分子激活剂和抑制剂是生物学上有用的，但受限于它们在体内的靶特异性和时空分辨率。最近出现的光遗传学策略可以补充化学/药理学方法。个体发生涉及将编码活性可被光活化的蛋白质的基因引入细胞和动物。光是一种独特的刺激，因为它可以以可逆的方式控制体内蛋白质的活性，并且具有化学物质无法达到的时空精度。细菌光敏色素类型的光感受器吸收近红外光，其具有上级组织穿透性质，因此允许来自不显眼的外部光源的蛋白质光活化。这对于整个动物（如小鼠）的研究尤其重要。最近，在“移植”天然光感受器模块以控制异源蛋白活性方面取得了进展。我们的长期目标是阐明工程原理的近红外光激活的蛋白质利用光敏模块的细菌光敏色素。这个探索性的建议将测试的假设，细菌光敏色素感光域可以激活不同的同源二聚体输出活动。我们将利用我们早期的研究BphG蛋白，一个独特的，近红外光激活的二鸟苷酸环化酶。核苷酸环化酶将用作工程化靶标。和一个执行者（效应器）半胱天冬酶合理设计细菌光敏色素为基础的蛋白质，我们将采用多管齐下的方法，涉及规避我们目前无法计算和结构分析的蛋白质与遗传筛选在E。杆菌工程近红外光激活的同源二聚体蛋白和细菌光敏色素工程的多学科方法的概念是创新和可行的，因为我们已经构建了第一个具有异源活性的光激活同源二聚体酶。本计画完成后，我们将进一步了解光在细菌光敏色素中的信号传递机制，并揭示构建同源二聚体近红外光活化蛋白的工程原理。由于大量信号蛋白作为同源二聚体起作用，光诱导的蛋白质同源二聚化可用于控制多种细胞功能，包括凋亡、分化、增殖、转化和粘附。这项研究意义重大，因为cAMP和cGMP控制许多细胞过程，包括生长，血糖水平，心脏收缩功能，学习，记忆和癌细胞存活。这里产生的光活化刽子手半胱天冬酶将允许研究人员使用温和和非侵入性的程序在整个动物中进行靶向细胞/组织杀伤。这些工具可能会在细胞生物学、免疫学和发育生物学中找到应用，并可能在癌症基因治疗中找到应用。