Experimental Strategies for Light-Induced Elimination of Protein Function in vivo

光诱导体内蛋白质功能消除的实验策略

• 批准号: 8623032
• 负责人: DAVID S. STEIN
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8623032
• 关键词: Address; Animal Model; Avena sativa; Biological; Biology; Brain; Caenorhabditis elegans; Cell Culture Techniques; Cell Cycle; Cell Survival; Cells; Cessation of life; Chimeric Proteins; Development; Disease; Dorsal; Dorsal-Ventral Pattern Formation; Double-Stranded RNA; Drosophila genus; Drosophila melanogaster; Embryo; Exposure to; Family; Future; Gene Expression; Gene Mutation; Gene Proteins; Generations; Genes; Genetic; Genetic Models; Goals; Individual; Investigation; Kinetics; Laboratory Organism; Life; Light; Mammals; Mediating; Methods; Modification; Mus; Mutation; Neurons; Oats; Organism; Pattern; Peptides; Phenotype; Physiology; Pilot Projects; Play; Process; Proteins; RNA Interference; Reagent; Role; Saccharomyces cerevisiae; Signal Transduction; Staging; System; Techniques; Technology; Temperature; Testing; Thale Cress Proteins; Time; Tissues; Transgenic Organisms; Ubiquitin; Ubiquitination; Work; Yeasts; Zebrafish; absorption; base; cryptochrome 2; design; fly; gene function; genetic analysis; in vivo; interest; loss of function; loss of function mutation; multicatalytic endopeptidase complex; mutant; optogenetics; phototropin; protein degradation; protein function; public health relevance; rapid technique; recognins; research study; temperature sensitive mutant; tool; ubiquitin ligase; yeast genetics; yeast protein

DESCRIPTION (provided by applicant): For genes encoding proteins that are required for organismal or cell viability, an inability to generate and examine loss-of-function mutant phenotypes can be an impediment to elucidating protein function. Homozygous mutant individuals may die at an early stage in development, making it difficult to establish the differen roles that the relevant proteins play at later stages of development. Tissue-specific generation of homozygous mutant clones, or expression of dsRNA, can in some cases overcome this limitation. However, these strategies are subject to the problem of protein perdurance beyond the time at which gene expression ceases, which can complicate analyses in which rapid elimination of gene function is required. Temperature-sensitive (ts) mutations provide an alternative means of conditionally eliminating gene product function. However, ts mutants are laborious to isolate and cannot be applied to the study of protein function in homeothermic organisms such as mammals. The objective of the investigations described in this proposal is the development of a general method for rapid, spatially- and temporally-controlled light-induced elimination of proteins of interest for phenotypic analysis. Two specific aims will be pursued to develop and test two distinct experimental methods to achieve protein elimination. Specific Aim 1 will utilize a small protein tag that we refer to as the photodegron, which can be expressed as a genetic fusion to other proteins. The photodegron undergoes a light-dependent conformational change that exposes a degradation signal recognized by a ubiquitous class of ubiquitin ligases, the N-recognins. In preliminary studies carried out in yeast, the photodegron mediated light-dependent elimination of function of the heterologous proteins to which it was attached. Continuing work will examine the kinetics of photodegron-induced degradation in yeast and test the ability of the photodegron to direct protein degradation in Drosophila embryos. Additional studies will increase the versatility of the photodegron method by incorporating a signal that targets it for degradation via the Nedd4 family of ubiquitin ligases. The investigations in Specifi Aim 2 will pursue a second strategy to achieve light- induced protein degradation in vivo by making use of the light-dependent interaction between the Arabidopsis thaliana proteins Cryptochrome 2 (CRY2) and CIB1. Planned experiments will test the ability of CRY2-ubiquitin ligase fusion proteins to mediate the degradation of proteins fused to CIB1 in Drosophila embryos. Based on recent advances in the understanding of light-responsive proteins and of ubiquitin/proteasome-mediated protein degradation, we hypothesize that it will be possible to use the sophisticated genetics of yeast and Drosophila to develop facile experimental strategies for rapid, temporally- and spatially-controlled protein elimination. These methods will overcome several limitations associated with currently available strategies for generating protein loss-of-function phenotypes and will provide a powerful tool for the study of medically important proteins involved in a wide variety of biological questions and experimental organisms.

描述（由申请人提供）：对于编码生物体或细胞活力所需的蛋白质的基因，不能产生和检查功能丧失突变体表型可能是阐明蛋白质功能的障碍。纯合突变个体可能在发育的早期阶段死亡，使得难以确定相关蛋白在发育后期发挥的促性腺激素作用。组织特异性生成 纯合突变体克隆或dsRNA的表达在某些情况下可以克服这种限制。然而，这些策略受到蛋白质持久性超过基因表达停止的时间的问题的影响，这可能使需要快速消除基因功能的分析复杂化。温度敏感性（ts）突变提供了有条件地消除基因产物功能的替代手段。然而，ts突变体的分离是费力的，并且不能应用于恒温生物如哺乳动物中的蛋白质功能的研究。本提案中所述研究的目的是开发一种通用方法，用于表型分析中感兴趣的蛋白质的快速、空间和时间控制的光诱导消除。将追求两个具体目标，以开发和测试两种不同的实验方法，以实现蛋白质消除。Specific Aim 1将利用我们称为光降解决定子的小蛋白质标签，其可以表达为与其他蛋白质的遗传融合。光降解决定子经历光依赖性构象变化，其暴露由普遍存在的一类泛素连接酶（N-识别蛋白）识别的降解信号。在酵母中进行的初步研究中，光降解决定子介导了其所连接的异源蛋白功能的光依赖性消除。继续的工作将研究酵母中光降解子诱导降解的动力学，并测试光降解子在果蝇胚胎中直接蛋白质降解的能力。进一步的研究将增加光降解决定子方法的多功能性，通过掺入一个信号，靶向它通过泛素连接酶的Nedd 4家族降解。Specifi Aim 2中的研究将采用第二种策略，通过利用拟南芥蛋白隐花色素2（Cryptochrome 2）和CIB 1之间的光依赖性相互作用来实现体内光诱导的蛋白质降解。计划的实验将测试β 2-泛素连接酶融合蛋白介导果蝇胚胎中与CIB 1融合的蛋白质降解的能力。基于对光响应蛋白和泛素/蛋白酶体介导的蛋白质降解的理解的最新进展，我们假设将有可能使用酵母和果蝇的复杂遗传学来开发快速，时间和空间控制的蛋白质消除的简便实验策略。这些方法将克服与目前可用的用于产生蛋白质功能丧失表型的策略相关的几个限制，并且将为研究涉及各种生物学问题和实验生物体的医学重要蛋白质提供有力的工具。