Metallothionein mediated metal trafficking in the model invertebrate C.elegans: dissection of structural and functional differentiation

自由生活的线虫（或线虫）秀丽隐杆线虫被广泛用作研究与生命有关的过程的模式生物。秀丽隐杆线虫的基因组已被完全测序，其基本的生物学机制在秀丽隐杆线虫中经常与人类相似，因此从秀丽隐杆线虫中获得的结果有助于提供对人类生物学和生理学的详细了解。像任何其他生物一样，秀丽隐杆线虫从环境中获取必需的营养，同时避免被有毒化合物毒害。然而，有些元素在低浓度下是必需的，但在高浓度时就有毒了。例如，重金属锌对几乎所有的生物过程都是必不可少的，锌缺乏已被证明对生殖、生长和发育有有害影响。同样，铜虽然在生物系统中的含量通常低于锌，但对许多生物过程同样重要，例如能量产生和软骨和结缔组织的形成。这两种重金属都需要严格控制，因为过量会产生剧毒。金属硫蛋白（MTs）家族是控制必需金属可利用水平的一个有前途的参与者，金属硫蛋白是存在于所有高等生命形式（植物、真菌和动物）中的小金属结合蛋白。然而，即使在哺乳动物mt被发现的50年后，其确切的生物学作用仍然是一个谜。一个持续存在的障碍是人类基因组中存在超过20种不同的基因，而小鼠基因组中至少有4种。因此，很难确定每个MT的同种异构体的特定功能。相比之下，秀丽隐杆线虫基因组的完全测序只有两个同种异构体。在一名申请者之前的工作中，研究表明这两种基因（单独或一起）可以降低对重金属中毒的易感性。此外，镉、铜和锌会触发蠕虫肠道细胞中mtl-1和mtl-2的表达。然而，mtl-1在咽部的任何时候都明显表达，这表明它是一种金属（可能是锌）传感器。一个特别有趣的点是两个基因和相应蛋白质之间的差异，即mtl-1在其c端有15个额外的残基，其中三个能够结合金属离子。我们建议使用尖端的分子生物学、遗传学和全动物生命周期参数来研究这两种蛋白的差异功能，特别关注15个c端残基。与此同时，我们将详尽地描述这两种蛋白质的金属结合特性，并确定它们的三维结构，因为我们相信生物分子及其功能只能通过对其结构和作用分子机制的详细了解来理解。这两个实验室都是这两个领域的专家，我们相信，通过我们的互动，我们将达到一个否则不可能获得的知识和理解水平。

The free-living roundworm (or nematode) C.elegans is extensively used as a model organism to study processes that are pertinent to life. Its genome has been fully sequenced, and basic biological mechanisms operating in C.elegans frequently parallel those in man, and thus results obtained from C.elegans have been instrumental in providing a detailed insight into human biology and physiology. As any other living organism, C.elegans obtains essential nutrients from the environment whilst avoiding being poisoned by noxious compounds. However, some elements are essential at low concentrations, but toxic at elevated levels. The heavy metal zinc, for example, is indispensable to virtually all biological processes, and zinc deficiency has been documented to have deleterious effects on reproduction, growth, and development. Similarly, copper, although usually present in biological systems at lower amounts than zinc, is equally essential to many biological processes, such as energy production and the formation of cartilage and connective tissue. Both heavy metals underlie a stringent control as excess amounts are highly toxic. A promising player in the control of available levels of essential metals is the family of Metallothioneins (MTs), small metal-binding proteins that are present in all higher life forms (plants, fungi and animals). However, even after a five decade spanning time period since its discovery, the precise biological role(s) of mammalian MTs remain enigmatic. One persisting hurdle is the presence of over 20 different genes within the human genome, and at least four in the genome of the mouse. In consequence, it has been very difficult to pin down an isoform specific function for each MT. In contrast, the fully sequenced C.elegans genome has only two isoforms. In previous work by one of the applicants, it was shown that both genes (individually or together) decrease the susceptibility towards heavy metal poisoning. In addition, cadmium, copper and zinc trigger the expression of both mtl-1 and mtl-2 in the cells of the worm's gut. However, mtl-1 is notably expressed at all times in the pharynx suggesting a role as a metal (possibly zinc) sensor. A particular point of interest is the difference between the two genes and the corresponding proteins, namely mtl-1's 15 additional residues in its C-terminus, three of which are capable of binding metal ions. We propose to use cutting-edge molecular biology, genetic and whole animal life-cycle parameters to study the differential functions of the two proteins with particular focus on the 15 C-terminal residues. In parallel, we will exhaustively characterise the metal binding properties of both proteins, and also determine their 3-dimensional structures, as we believe that biomolecules and their function can only be understood with a detailed knowledge of their structure and the molecular mechanisms of their action. The two labs are experts in either of the two areas, and we are convinced that through our interaction we will achieve a level of knowledge and understanding that would otherwise be impossible to obtain.