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）：以下内容包含M. Gomelsky要求不释放给政府以外人员的专有/特权信息，除了审查和评估目的外。 项目摘要：信号转导途径的小分子激活剂和抑制剂在生物学上有用，但受其靶特异性和体内时空分辨率的限制。最近出现的光遗传策略可以补充化学/药理方法。个体发育涉及将可以光活化活性的蛋白质的细胞和动物引入基因和动物。光是一种独特的刺激，它可以以可逆的方式在体内控制蛋白质活性，并且具有化学物质无法实现的时空精度。细菌性色素类型的感光体吸收近红外光，其具有较高的组织穿透特性，从而允许蛋白质光活化来自不引人注目的外部光源。这对于对整个动物（例如小鼠）的研究尤其重要。最近，在“移植”天然光感受器模块中取得了进展，以控制异源蛋白活性。我们的长期目标是使用细菌色素的光感模块阐明工程近红外光活化蛋白的原理。该探索性提案将检验以下假设：拟菌色素光感受器结构域可以激活各种同型二聚体输出活性。我们将利用对BPHG蛋白的早期研究，BPHG蛋白是一种独特的，近红外的光活化的Diguanyll Cyclase。核苷酸循环酶将用作工程目标。 execution子（效应子）caspase理性地设计基于细菌植物色素的蛋白质，我们将采用一种多头方法，涉及我们目前对大肠杆菌中基因筛查的蛋白质计算和结构分析的无能为力。提议的工程近红外光激活同型蛋白和细菌性植物色素工程的多学科方法的概念具有创新性和可行性，因为我们已经构建了具有异源活性的第一个光活化同型含量酶。该项目完成后，我们预计将提高我们对拟植物色素中光诱导的信号传播机制的理解，并发现用于构建同型二聚体近红外光活化蛋白的工程原理。由于大量信号蛋白充当均二聚体，因此可以使用光诱导的蛋白均二聚化来控制各种细胞功能，包括细胞凋亡，分化，增殖，转化和粘附。这项研究很重要，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，记忆和癌细胞存活。此处生成的光活化的execution子caspase将使研究人员能够使用轻度和无创方法在整个动物中进行靶向细胞/组织杀死。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，并有可能在癌症基因治疗中进行应用。 公共卫生相关性：拟议的研究与公共卫生有关，因为对保守信号通路的功能和机制的了解对于我们对正常和疾病状态的理解至关重要。环状核苷酸，CAMP和CGMP是影响各种细胞功能的通用第二使者。在具有较高空间和时间分辨率的动物模型中控制第二使者的能力具有我们对肿瘤发生，心血管功能，糖尿病发展和神经系统疾病的理解的潜力。这里开发的胱天蛋白酶将使研究人员能够使用轻度和无创治疗的活动物中进行靶向细胞杀死。因此，拟议的研究在细胞生物学，免疫学和发育生物学中的应用直接解决了NIH的使命。这些工具可能会发现癌症基因疗法。