Novel Molecules as In Vivo Biological Probes

作为体内生物探针的新型分子

基本信息

批准号：
9330164
负责人：
THOMAS James WANDLESS
金额：
$ 32.1万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9330164
关键词：
Affinity Alpha Cell Avena sativa Behavior Bilirubin Binding Binding Sites Biological Biological Models Cell physiology Cells Chimeric Proteins DNA Dependence Development Dose Engineering Ensure Equilibrium Eukaryotic Cell Family Fluorescence Genes Genetic Genetic Transcription Goals Human Engineering Ligand Binding Ligands Light Malaria Mammalian Cell Mammals Mediating Methodology Methods Modeling Optical Methods Parasites Permeability Pharmaceutical Preparations Plasmodium falciparum Proteins Quality Control RNA Interference Research Research Personnel Resolution Specificity System Tacrolimus Binding Protein 1A Tacrolimus Binding Proteins Techniques Technology Tertiary Protein Structure Testing Toxoplasma gondii Work Yeasts cofactor design falls human disease improved in vivo interest knock-down mRNA Precursor misfolded protein multicatalytic endopeptidase complex mutant novel overexpression programs protein aminoacid sequence protein degradation protein function public health relevance small molecule

项目摘要

DESCRIPTION: The broad, long-term objective of this research program is to develop general technology to conditionally regulate protein function at the level of the protein molecules rather than by targeting the DNA or mRNA precursors that encode a protein-of-interest. This technology is highly specific for the targeted protein and provides rapid and tunable control of protein function using cell-permeable small molecules or non-toxic light. The goal is to engineer small protein domains called destabilizing domains that are rapidly degraded when expressed in mammalian cells. The instability of destabilizing domains is faithfully conferred to other proteins fused to these small domains, allowing researchers to predictably control the levels of any protein-of-interest. One specific aim of this research program will result in new destabilizing domains that are intrinsically fluorescent, allowing researchers to quantify protein levels using optical methods. A second aim of this research is to produce destabilizing domains that are regulated by blue light rather than small molecules, thus enabling the control of protein stability with high spatial resolution. A third aim of these studies will provide a family of destabilizing domains whose cellular levels can be raised by treatment with one ligand or whose cellular levels can be made to fall by treatment with a different ligand. In this case a single destabilizin domain could be used to test the effects of overexpression with one ligand or strong knock-down with a different ligand in an isogenic background. The fourth aim of this research program is to produce destabilizing domains for use in Apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii. A mechanistic understanding of how these domains are recognized and degraded in mammalian cells will make this technology more useful to users. These studies may also reveal general mechanisms that cells use to recognize and degrade unfolded or misfolded proteins, and these mechanisms are likely relevant to human diseases.

描述：该研究计划的广泛长期目标是开发一般技术以有条件地调节蛋白质分子水平的蛋白质功能，而不是针对编码蛋白质利益蛋白的DNA或mRNA前体。该技术对靶向蛋白质高度特异，并使用可渗透细胞的小分子或无毒光对蛋白质功能进行快速可调的控制。目的是设计称为不稳定结构域的小蛋白质结构域，这些结构域在哺乳动物细胞中表达时会迅速降解。破坏稳定域的不稳定性忠实地赋予了其他蛋白质与这些小领域融合在一起，使研究人员可以预测控制任何利益蛋白质的水平。该研究计划的一个具体目的将导致新的破坏稳定的域，这些域本质上是荧光的，从而使研究人员可以使用光学方法来量化蛋白质水平。这项研究的第二个目的是产生由蓝光而不是小分子调节的不稳定域，从而可以控制蛋白质稳定性具有高空间分辨率。这些研究的第三个目的将提供一个破坏稳定的结构域的家族，其细胞水平可以通过使用一个配体或可以通过使用不同的配体治疗的细胞水平降低的细胞水平提高。在这种情况下，单个DESTABILIZIN结构域可用于测试用一个配体或强烈的敲低的敲低表达的效果，并在等源性背景下具有不同的配体。该研究计划的第四个目的是生产不稳定的域，用于用于apicomplexan寄生虫，例如恶性疟原虫和弓形虫弓形虫。对哺乳动物细胞中如何识别和降解这些域的机械理解将使该技术对用户更有用。这些研究还可能揭示了细胞用来识别和降解未折叠或错误折叠蛋白的一般机制，这些机制可能与人类疾病有关。