Novel Molecules as In Vivo Biological Probes

描述：这项研究计划的广泛、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，而不是通过瞄准编码目标蛋白质的DNA或mRNA前体。这项技术对目标蛋白质具有高度特异性，并使用细胞渗透性小分子或无毒光线提供对蛋白质功能的快速和可调控制。其目标是设计被称为不稳定结构域的小蛋白结构域，当在哺乳动物细胞中表达时，这些结构域会迅速降解。不稳定结构域的不稳定性被忠实地赋予其他蛋白质 融合到这些小区域，使研究人员能够预测地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不稳定结构域，这些结构域本质上是荧光的，使研究人员能够使用光学方法来量化蛋白质水平。这项研究的第二个目标是产生受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。 具有高空间分辨率。这些研究的第三个目标将提供一系列不稳定结构域，这些结构域的细胞水平可以通过使用一种配体来提高，或者其细胞水平可以通过使用不同的配体来降低。在这种情况下，可以使用一个单独的去稳定素域来测试在相同的基因背景下，用一个配体过度表达或用不同的配体强烈敲除的效果。这项研究计划的第四个目标是产生不稳定结构域，用于恶性疟原虫和弓形虫等Apicomplexan寄生虫。从机制上理解这些结构域在哺乳动物细胞中是如何识别和降解的，将使这项技术对用户更有用。这些研究还可能揭示细胞用来识别和降解未折叠或错误折叠的蛋白质的一般机制，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.