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

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.

The two C. elegans metallothionein (MT) isoforms show intriguing differences between their primary structures and their expression pattern: Although mtl-1 and mtl-2 share 62% sequence identity, and their expression in the gut can be induced by external heavy metals (Cd, Cu, Zn), mtl-1 has a 15 amino-acid C-terminal extension with 3 additional potential metal-binding residues. Most remarkable is the finding that only mtl-1 is constitutively expressed in the lower bulb of the pharynx. Our recent investigations have shown that, besides a role in Cd detoxification, the MTs are likely to have more general roles in metal ion homeostasis. We propose to determine these functions in detail, and carry out a comprehensive in vitro characterisation of the biophysical and biochemical properties of these two MTs, to advance knowledge of the relation between structure, solution properties, and function of metallothioneins. In addition, we will pay particular attention to the significance of the constitutive expression and C-terminal extension in mtl-1. Functional characterisation will be achieved by phenotyping C. elegans strains which over-express mtl-1 and mtl-2, by studying the impact of Zn and Cu depletion on a mtl-null strain, and by studying strains expressing mtls in which the C-termini have been mutated. In addition. all strains created will be evaluated in response to their susceptibility towards paraquat induced oxidative stress. We will also explore gene regulation and the genetic interaction network of mtl-1 and mtl-2 using customised micro-arrays. Biophysical and biochemical studies will be carried out on recombinantly (E. coli) expressed and purified proteins, and will include full structure determinations by state-of-the-art solution NMR methods. Metal ion binding thermodynamics and kinetics will be determined using multinuclear (1H, 19F and 111Cd) NMR spectroscopy, high-resolution electrospray mass spectrometry, ICP-MS and absorption spectroscopies.