Cell-specific interactions of snail Cu metallothionein with hemocyanin: A metallomic approach

金属硫蛋白(Metals thioneins，MTS)是一类富含半胱氨酸的金属结合蛋白，对过渡金属离子具有高亲和力。它们使有毒金属失活，并有助于维持细胞内必需微量元素的动态平衡和分布。在陆生蜗牛中，铜特异的金属硫蛋白亚型(CuMT)在所谓的红血球中表达。这些是在软体动物中发现的特殊细胞。红细胞也是血蓝蛋白的表达部位，血蓝蛋白是蜗牛携带铜的呼吸蛋白。CuMT与呼吸铜蛋白的细胞特异性共表达表明，在这些动物中，CuMT可能是新生HC分子的铜供体。这一发现得到了以下发现的支持：在节肢动物中，特定的MT亚型也可以将铜输送到节肢动物的HCS中。在目前的提案中，我们希望通过应用金属组学方法在两种蜗牛物种Helix pomatia和Cantareus assus中检验这一假说。为此，我们组建了一支研究人员网络团队，他们的专业知识和技能集中在毒理学、动物生理学、细胞生物学、生物化学和生物无机化学、基因组学、生物信息学和分子生物学等领域。研究活动将按照致力于CuMT、铜结合蛋白和配体以及蜗牛HC之间金属相互作用的不同方面的四个工作包(WP)来组织。(1)在WP 1中，我们将生成基因组和转录数据库，以筛选与我们的建议相关的蛋白质的存在(这两个物种中存在所有MT和HC亚型，以及可能参与铜向新生HC分子转移的其他潜在蛋白质候选)。(2)在WP 2中，我们将利用铜同位素盐进行从钉螺CuMT到HC的铜转移实验，通过(I)在自然分离的蜗牛CuMT和apo-HC混合物中进行体外交换实验，以及(Ii)在体内暴露于铜同位素盐的大肠杆菌细胞中重组表达CuMT和HC。(3)在WP 3中，我们将应用酵母双杂交方法筛选蜗牛CuMT和HC之间潜在的蛋白质-蛋白质相互作用。(4)在WP 4中，我们将通过成像方法显示潜在的CuMT-HC相互作用，使用(I)透射电子显微镜和ESI/EELS筛选铜在红细胞室的分布，(Ii)LSM用于CuMT发光，CuMT和HC mRNA荧光原位杂交，以及(Iii)透射电子显微镜和Western blotting用于CuMT/HC共定位。我们希望从我们的研究中更好地理解铜如何被分配到一个重要的含铜无脊椎动物蛋白质中，可能是通过CuMT的参与。一个特别耐人寻味，但也是不可否认的推测的前景是，可能会发现蜗牛CuMT的一种伴侣功能(因此也是类似酶的功能)。如果这是真的，这一发现将极大地改变我们对MTS整体功能意义的看法。

Metallothioneins (MTs) constitute a family of cysteine-rich metal binding proteins with a high affinity for transition metal ions. They inactivate toxic metals and help to maintain homeostatic balance and distribution of essential trace elements within the cell. In terrestrial snails, a Cu-specific MT isoform (CuMT) is expressed in the so-called rhogocytes. These are particular cells found within Mollusca. Rhogocytes are also the expression sites of hemocyanins (Hcs), the snails` Cu-carrying respiratory proteins. The cell-specific coexpression of CuMT with a respiratory Cu-protein suggests that in these animals, CuMT may serve as a Cu donator to nascent Hc molecules. This is supported by findings that in arthropods too, Cuspecific MT isoforms can deliver Cu to arthropod Hcs. In the present proposal, we want to test this hypothesis in the two snail species Helix pomatia and Cantareus aspersus by applying a metallomics approach. To this aim, we have assembled a networking team of researchers whose combined expertise and skills lie in the fields of toxicology, animal physiology, cell biology, biochemistry and bioinorganic chemistry, genomics, bioinformatics and molecular biology. The research activities will be structured along four work packages (WP) dedicated to different aspects of metal interactions between CuMT, Cu binding proteins and ligands, and snail Hc. (1) In WP 1, we will generate genomic and transcriptomic data bases to be screened for the presence of proteins relevant to our proposal (with all MT and Hc isoforms present in these two species, and with other potential protein candidates that may be involved in Cu transfer to nascent Hc molecules). (2) In WP 2, we will perform Cu transfer experiments from snail CuMT to Hc using Cu isotope salts, by performing (i) exchange experiments in natively isolated snail CuMT and apo-Hc mixtures in vitro, and (ii) recombinant expression of CuMT and Hc in E. coli cells exposed to Cu isotope salts in vivo. (3) In WP 3 we will screen for potential protein-protein interactions between snail CuMT and Hc by applying a yeast two-hybrid approach. (4) In WP 4, we will visualize potential CuMT-Hc interactions by imaging methods, using (i) TEM and ESI / EELS to screen for Cu distribution in rhogocyte compartments, (ii) LSM for CuMT luminescence, CuMT and Hc mRNA fluorescence in situ hybridization, and (iii) TEM and Western blotting for CuMT / Hc co-localization. We expect from our studies a better understanding of how Cu may be allocated to an important Cu-containing invertebrate protein, possibly by the involvement of a CuMT. A particularly intriguing, yet admittedly speculative, prospect would be the potential discovery of a chaperonin (and thus enzyme-like) function of snail CuMT. If true, this finding would dramatically change our view on the functional significance of MTs overall.