Near-infrared light activated protein photoswitches

近红外光激活蛋白质光开关

• 批准号: 8286092
• 负责人: Mark Gomelsky
• 金额: $ 18.21万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8286092
• 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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because knowledge of functions and mechanisms of conserved signaling pathways is essential for our understanding of normal and disease states. Cyclic nucleotides, cAMP and cGMP, are universal second messengers that affect a variety of cellular functions. The ability to control second messengers in animal models with high spatial and temporal resolution has the potential to bring our understanding of tumorogenesis, cardiovascular function, development of diabetes, and neurological disorders to previously unattained levels. Caspases developed here will allow researchers to perform targeted cell killing in live animals using a mild and noninvasive treatment. Applications in cell biology, immunology and developmental biology, thus, the proposed research directly addresses the NIH's mission. And potentially in cancer gene therapy these tools will likely find.

描述(由申请人提供)：以下包含M.Gomelsky要求不向政府以外的人发布的专有/特权信息，除非是出于审查和评估的目的。 小分子信号转导通路激活剂和抑制物在生物学上是有用的，但在体内受到靶标特异性和时空分辨率的限制。最近出现的一种光遗传策略可以补充化学/药理学方法。个体发育涉及将编码蛋白质的基因导入细胞和动物，这些蛋白质的活性可以被光激活。光是一种独特的刺激，因为它可以以一种可逆的方式控制体内的蛋白质活动，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，具有优越的组织穿透特性，从而允许蛋白质从不显眼的外部光源进行光激活。这对于对整个动物的研究尤其重要，比如小鼠。最近，在“移植”天然光感受器模块以控制异源蛋白质活性方面取得了进展。我们的长期目标是阐明利用细菌藻红素的光敏模块来改造近红外光激活蛋白质的原理。这一探索性的建议将检验细菌光敏素光受体结构域可以激活不同的同源二聚体输出活性的假设。我们将利用我们早期对BphG蛋白的研究，BphG蛋白是一种独特的近红外光激活的二鸟酰环化酶。核苷酸环化酶将被用作工程靶标。和执行者(效应者)caspase合理设计基于细菌光敏色素的蛋白质，我们将采用多管齐下的方法，包括绕过我们目前无法在大肠杆菌中进行基因筛选的蛋白质的计算和结构分析。提出的近红外光活化同源二聚体蛋白工程概念和细菌光敏色素工程的多学科方法是创新和可行的，因为我们已经构建了第一个具有异源活性的光活化同源二聚体酶。该项目完成后，我们将进一步了解细菌光敏色素的光诱导信号传播机制，并揭示构建同源二聚体近红外光激活蛋白的工程原理。由于大量的信号蛋白作为同源二聚体发挥作用，光诱导的蛋白质同源二聚可用于调控细胞的多种功能，包括细胞的凋亡、分化、增殖、转化和黏附。这项研究意义重大，因为cAMP和cGMP控制着许多细胞过程，包括生长、血糖水平、心脏收缩功能以及学习、记忆和癌细胞生存。这里产生的光激活的刽子手caspase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有针对性的细胞/组织杀戮。这些工具可能会在细胞生物学、免疫学和发育生物学中得到应用，并有可能在癌症基因治疗中得到应用。 与公共健康相关：拟议的研究与公共健康相关，因为对保守信号通路的功能和机制的了解对于我们理解正常和疾病状态是必不可少的。环核苷酸，cAMP和cGMP，是影响多种细胞功能的普遍的第二信使。在动物模型中以高空间和时间分辨率控制第二信使的能力有可能将我们对肿瘤发生、心血管功能、糖尿病发展和神经疾病的了解提高到前所未有的水平。这里开发的半胱氨酸天冬氨酸酶将允许研究人员使用温和和非侵入性的治疗方法在活体动物中进行靶向细胞杀伤。在细胞生物学、免疫学和发育生物学中的应用，因此，拟议的研究直接满足了NIH的使命。在癌症基因治疗中，这些工具很可能会找到。