Novel Molecules as In Vivo Biological Probes

产品说明：该研究计划的广泛，长期目标是开发通用技术，在蛋白质分子水平上有条件地调节蛋白质功能，而不是通过靶向编码感兴趣蛋白质的DNA或mRNA前体。该技术对靶蛋白具有高度特异性，并使用细胞可渗透的小分子或无毒光对蛋白质功能进行快速和可调的控制。其目标是设计称为去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迅速降解。不稳定结构域的不稳定性被忠实地赋予其他蛋白质 融合到这些小结构域，使研究人员能够预测控制任何感兴趣的蛋白质的水平。这项研究计划的一个具体目标是产生新的固有荧光的去稳定结构域，使研究人员能够使用光学方法定量蛋白质水平。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结构域，从而能够控制蛋白质的稳定性。 高空间分辨率。这些研究的第三个目的是提供一个去稳定化结构域家族，其细胞水平可以通过用一种配体处理而升高，或者其细胞水平可以通过用不同配体处理而降低。在这种情况下，可以使用单个去稳定化结构域来测试在同基因背景中用一种配体过表达或用不同配体强敲低的效果。该研究计划的第四个目的是产生用于顶复门寄生虫如恶性疟原虫和刚地弓形虫的去稳定结构域。对这些结构域如何在哺乳动物细胞中被识别和降解的机械理解将使这项技术对用户更有用。这些研究还可能揭示细胞用于识别和降解未折叠或错误折叠蛋白质的一般机制，这些机制可能与人类疾病有关。

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.