Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)4(CysS)11 cluster and a M(II)3(CysS)9 cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn7MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn7MT-1/Zn7MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn7MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn7MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn7MT-3 and the formation of the Pt-S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The 113Cd NMR studies of Pt1 113Cd6MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn7MT-3 were much faster than those of cisplatin and transplatin with Zn7MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn7MT-3 than Zn7MT-2 in the acquired resistance to platinum-based drugs are discusse

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

<p>Abstract</p> <p>Background</p> <p>FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006).</p> <p>Results</p> <p>We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1.</p> <p>Conclusion</p> <p>The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.</p

Reactivity of an antimetastatic organometallic ruthenium compound with metallothionein-2: relevance to the mechanism of action

The reaction of metallothionein-2 (MT-2) with the organometallic antitumour compound [Ru(eta(6)-p-cymene)Cl-2(pta)], RAPTA-C, was investigated using ESI MS and ICP AES. The studies were performed in comparison to cisplatin and significant differences in the binding of the two complexes were observed. RAPTA-C forms monoadducts with MT-2, at variance with cisplatin, that has been observed to form up to four adducts. These data, combined with ICP AES analysis, show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of zinc, suggesting that Cys residues are the target binding sites for the two metallodrugs. The competitive binding of RAPTA-C and cisplatin towards a mixture of ubiquitin (Ub) and MT-2 was also studied, showing that MT-2 can abstract RAPTA-C from Ub more efficiently than it can abstract cisplatin. The mechanistic implications of these results are discussed