权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

基本信息

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
项目类别：
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

项目摘要

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(申请人提供)：对于编码生物体或细胞生存所需的蛋白质的基因，无法产生和检查功能丧失的突变表型可能是阐明蛋白质功能的障碍。纯合突变个体可能会在发育的早期阶段死亡，这使得很难确定相关蛋白质在发育后期所扮演的不同角色。特定于组织的一代纯合子突变克隆或dsRNA的表达在某些情况下可以克服这一限制。然而，这些策略受到蛋白质持续时间超过基因表达停止的问题的影响，这可能会使需要快速消除基因功能的分析复杂化。温度敏感型(Ts)突变提供了一种有条件地消除基因产物功能的替代方法。然而，ts突变体很难分离，并且不能用于研究哺乳动物等恒温生物的蛋白质功能。这项建议中描述的研究的目标是开发一种通用的方法，用于快速、空间和时间控制的光诱导消除感兴趣的蛋白质，用于表型分析。将追求两个具体目标来开发和测试实现蛋白质消除的两种不同的实验方法。特殊目的1将利用一个小的蛋白质标签，我们称之为光降解子，它可以表达为与其他蛋白质的遗传融合。光降解蛋白经历光依赖的构象变化，暴露出一种普遍存在的泛素连接酶--N-识别素识别的降解信号。在酵母中进行的初步研究中，光降解子介导了与其连接的异源蛋白功能的光依赖消除。继续的工作将研究光降解子在酵母中诱导降解的动力学，并测试光降解子在果蝇胚胎中直接降解蛋白质的能力。其他研究将增加光降解方法的通用性，方法是加入一个信号，通过Nedd4泛素连接酶家族进行降解。在特定目标2中的研究将寻求第二种策略，通过利用拟南芥隐花色素2(CRY2)和CIB1之间的光依赖相互作用来实现体内光诱导的蛋白质降解。计划中的实验将测试CRY2-泛素连接酶融合蛋白在果蝇胚胎中介导CIB1融合蛋白降解的能力。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，我们假设有可能利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单实验策略。这些方法将克服与目前可用的产生蛋白质功能丧失表型的策略相关的几个限制，并将为研究涉及广泛的生物学问题和实验生物体的具有医学重要性的蛋白质提供强有力的工具。