Near-infrared light activated protein photoswitches

近红外光激活蛋白质光开关

基本信息

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
项目类别：
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

项目摘要

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 要求不得向政府以外人员透露的专有/特权信息，除非用于审查和评估目的。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是有用的，但受到其靶标特异性和体内时空分辨率的限制。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其活性可被光激活的蛋白质的基因引入细胞和动物中。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，具有优异的组织穿透特性，从而允许不引人注目的外部光源对蛋白质进行光活化。这对于对整个动物（例如小鼠）的研究尤其重要。最近，“移植”天然光感受器模块来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使用细菌光敏色素的光敏模块工程近红外光激活蛋白质的原理。这一探索性提议将检验细菌光敏色素光感受器结构域可以激活多种同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苷酸环化酶。核苷酸环化酶将用作工程靶标。刽子手（效应器）半胱天冬酶合理地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，包括克服我们目前无法通过大肠杆菌基因筛选对蛋白质进行计算和结构分析的情况。所提出的工程近红外光激活同二聚蛋白的概念和细菌光敏色素工程的多学科方法是创新且可行的，因为我们已经构建了第一个具有异源活性的光激活同二聚酶。该项目完成后，我们预计将加深对光敏色素中光诱导信号传播机制的理解，并揭示构建同型二聚体近红外光激活蛋白的工程原理。由于大量信号蛋白以同型二聚体的形式发挥作用，光诱导的蛋白质同型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、心脏收缩功能以及学习、记忆和癌细胞存活。这里产生的光激活刽子手半胱天冬酶将使研究人员能够使用温和且非侵入性的程序对整个动物进行靶向细胞/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得到应用，并可能在癌症基因治疗中得到应用。公共健康相关性：拟议的研究与公共健康相关，因为了解保守信号通路的功能和机制对于我们理解正常和疾病状态至关重要。环核苷酸、cAMP 和 cGMP 是影响多种细胞功能的通用第二信使。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使我们对肿瘤发生、心血管功能、糖尿病发展和神经系统疾病的理解达到以前未达到的水平。这里开发的半胱天冬酶将使研究人员能够使用温和、无创的治疗方法对活体动物进行靶向细胞杀伤。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。这些工具可能会在癌症基因治疗中找到应用。