Structure, Function and Transcriptional Regulation of a Copper-Specific Metallothionein in Callinectes sapidus

摘要建议MCB-0080075PI：有机体的各种功能依赖于铁和铜等痕量金属。然而，当这些金属过量时，它们可能会产生有毒影响。这项研究项目的目的是加深我们对生物体使用的分子策略的理解，这些策略将潜在的有毒铜转化为对生物有用的形式，同时避免其毒副作用。许多生物使用金属特有的蛋白质，称为金属硫蛋白，用于有毒金属的解毒，如镉和汞，以及用于解毒多余的必需金属，如铜。这个项目使用了蓝蟹，这种生物的铜代谢非常活跃，与血蓝蛋白的合成和降解有关，血蓝蛋白是一种依赖铜的蛋白质，动物用来将氧气从鳃输送到组织中。早期的研究发现，螃蟹的金属硫蛋白受锌控制，就像脊椎动物一样，但也有一种铜特有的金属硫蛋白，其合成受铜而不是锌控制。本研究项目有三个目标。首先，将检验铜特定的金属硫蛋白可以提供生物合成血蓝蛋白所需的铜的假设。这些研究可能揭示这个金属硫蛋白家族的新成员的新的生物学功能。其次，由于在分子水平上了解蛋白质的功能需要了解其结构，因此将确定金属硫蛋白蛋白质的三维结构。这些研究将提供高等生物体中铜金属硫蛋白的第一个结构。第三，研究铜-金属硫蛋白基因的转录调控。这项工作可能导致首次在高等生物体中展示铜敏感转录因子，并可能为进一步研究高等生物中铜敏感和信号转导提供基础。

ABSTRACT Proposal MCB-0080075PI: Marius BrouwerOrganisms are dependent on trace metals such as iron and copper for various functions. However, when these metals are in excess they may have toxic effects. The goal of this research project is to further our understanding of the molecular strategies used by organisms to convert potentially toxic copper into a biologically useful form, while avoiding its toxic side effects. Many organisms use metal-specific proteins, called metallothioneins, for detoxification of toxic metals, such as cadmium and mercury, and for detoxification of excess essential metals, such as copper. This project uses the blue crab, an organism that has a very active copper metabolism associated with the synthesis and degradation of hemocyanin, a copper-dependent protein used by the animal to carry oxygen from the gills to the tissues. Earlier work has found that crabs have metallothioneins that are controlled by zinc, as in vertebrates, but also have a copper-specific metallothionein whose synthesis is controlled by copper and not by zinc. This research project has three objectives. First, the hypothesis that the copper-specific metallothionein can provide copper that is required for the biosynthesis of hemocyanin will be examined. These studies may reveal a novel biological function for this new member of the metallothionein family. Secondly, since understanding of the function of a protein at the molecular level requires knowledge of its structure, the three-dimensional structure of the metallothionein protein will be determined. These studies will provide the first structure of a copper-metallothionein of a higher organism. Thirdly, the transcriptional control of the copper-metallothionein gene will be studied. This work may result in the first demonstration of a copper-sensing transcription factor in a higher organism, and may provide the foundation upon which further studies on copper sensing and signaling in higher organisms may be based.