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

自由生活的蛔虫（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。它的基因组已被完全测序，在秀丽隐杆线虫中运作的基本生物学机制经常与人类相似，因此从秀丽隐杆线虫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。与其他生物一样，秀丽隐杆线虫从环境中获得必需的营养物质，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，但在高浓度下是有毒的。例如，重金属锌对几乎所有生物过程都是必不可少的，并且锌缺乏已被证明对生殖、生长和发育具有有害影响。类似地，铜虽然通常以低于锌的量存在于生物系统中，但对许多生物过程同样重要，例如能量产生以及软骨和结缔组织的形成。这两种重金属都是严格控制的基础，因为过量是剧毒的。在控制必需金属的可用水平方面，一个有前途的参与者是金属硫蛋白（MT）家族，这是一种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，即使在自其发现以来的五十年跨越时间段之后，哺乳动物MT的精确生物学作用仍然是谜。一个持续存在的障碍是人类基因组中存在20多个不同的基因，小鼠基因组中至少有4个。因此，很难确定每个MT的同种型特异性功能。相比之下，完全测序的秀丽隐杆线虫基因组只有两种亚型。在申请人之一的先前工作中，表明两种基因（单独或一起）降低了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在咽部中始终显著表达，表明其作为金属（可能是锌）传感器的作用。特别感兴趣的是两个基因和相应蛋白质之间的差异，即mtl-1在其C-末端的15个额外残基，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，遗传学和整个动物生命周期的参数来研究这两种蛋白质的差异功能，特别关注15个C-末端残基。与此同时，我们将详尽地分析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详细了解它们的结构及其作用的分子机制来理解。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，我们将达到一个知识和理解的水平，否则是不可能获得的。

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.