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

金属硫蛋白（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。它们灭活有毒金属，并有助于维持细胞内必需微量元素的稳态平衡和分布。在陆生蜗牛中，cu特异性MT异构体（CuMT）在所谓的球状细胞中表达。这些是在软体动物体内发现的特殊细胞。血红细胞也是蜗牛携带铜的呼吸蛋白——血青素（Hcs）的表达位点。CuMT与呼吸Cu蛋白的细胞特异性共表达表明，在这些动物中，CuMT可能作为新生Hc分子的Cu供体。节肢动物的研究结果也支持了这一点，特异性MT亚型可以将Cu传递到节肢动物的Hcs。在本提案中，我们希望通过应用金属组学方法在两种蜗牛物种螺旋pomatia和Cantareus aspersus中验证这一假设。为此，我们组建了一个由毒理学、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的研究人员组成的网络团队。研究活动将按照四个工作包（WP）进行组织，专门研究CuMT、Cu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。(1)在WP 1中，我们将生成基因组和转录组数据库，以筛选与我们的建议相关的蛋白质（在这两个物种中存在所有MT和Hc同种异构体，以及其他可能参与Cu转移到新生Hc分子的潜在候选蛋白质）。(2)在WP 2中，我们将使用Cu同位素盐进行Cu从蜗牛CuMT到Hc的转移实验，通过(i)在体外进行天然分离的蜗牛CuMT和载脂蛋白Hc混合物的交换实验，以及（ii）在体内暴露于Cu同位素盐的大肠杆菌细胞中重组CuMT和Hc的表达。(3)在WP 3中，我们将通过酵母双杂交方法筛选蜗牛CuMT和Hc之间潜在的蛋白质相互作用。(4)在WP 4中，我们将通过成像方法可视化潜在的CuMT-Hc相互作用，使用(i) TEM和ESI / EELS筛选铜在红细胞区室中的分布，（ii） LSM用于CuMT发光，CuMT和Hc mRNA荧光原位杂交，以及（iii） TEM和Western blotting用于CuMT / Hc共定位。我们期望从我们的研究中更好地了解铜是如何分配到一个重要的含铜无脊椎动物蛋白上的，可能是通过一个CuMT的参与。一个特别有趣的，但不可否认的推测，前景将是潜在的发现伴侣蛋白（因此类似酶）的蜗牛CuMT的功能。如果这是真的，这一发现将极大地改变我们对mt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.