The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four.65 Znlabeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.The possible functional implications of the zinc cluster structure of metallothionein (MT) have been the subject of much speculation (1-3). In contrast with other zinc proteins, MT is remarkable in that it binds zinc with high thermodynamic stability [K d ϭ 1.4 ϫ 10 Ϫ13 M at pH 7.0 for human MT (4)] while exhibiting a kinetic lability that results in facile zinc exchange reactions (5). This unusual combination appears to be a characteristic property of the clusters and, likely, a critical element in their hypothetical function to ''[provide] zinc where and when needed and for whatever role'' (6). Thus, in MT the protein plays a role in the biological function of zinc, a paradigm quite different from that in most other zinc proteins where zinc plays a role in the biological function of the protein. The tight binding of zinc to MT raises questions of how it is released and whether or not the release is controlled. In this regard, we have identified glutathione disulfide (GSSG) as a cellular ligand that reacts with MT and mobilizes zinc, resulting in the suggestion that the zinc content of MT is linked to the redox state of glutathione in the cell in such a manner that zinc remains bound to MT as long as high thiol reducing power prevails and is released once the redox balance becomes more oxidizing (7). We here extend these findings by studying the role of glutathione-mediated zinc release in the presence of a zinc acceptor such as an apoenzyme. In particular, we show that only one of the seven zinc atoms i...

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Synergistic therapy that combines chemo-, gene-, or photothermal means shows great potential for enhancing the therapeutic effects on cancers. Tumor-targeted nanoparticles based on a doxorubicin (DOX)-gated mesoporous silica nanocore (MSN) encapsulated with permeability glycoprotein (P-gp) small interfering RNA (siRNA) and a polydopamine (PDA) outer layer for DOX loading and folic acid decoration are designed. The multifunctional nanoplatform tactfully integrates chemo-(DOX), gene-(P-gp siRNA), and photothermal (PDA layer) substances in one system. In vitro results reveal that DOX release behaviors are both pH-and thermal-responsive and the release of co-delivered P-gp siRNA is also pH-dependent due to the pH-cleavable DOX gatekeeper on MSN. In addition, due to the near-infrared light-responsive PDA outer layer and folic acid conjugation, the nanoparticles exhibit outstanding photothermal activity and selective cell targeting ability. Subsequently, in vitro and in vivo antitumor experiments both demonstrate the enhanced antitumor efficacy of the multifunctional nanoparticles, indicating the significance of synergistic therapy combining chemo-, gene-, and photothermal treatments in one system. Cancer Therapy

The ␣-and ␤-polypeptides of human metallothionein (isoform 2), obtained by chemical synthesis, were converted into their respective zinc͞thiolate clusters, and each domain was investigated separately. Proton titration data for the N-terminal ␤-domain fit a simple model with three ionizations of the same apparent pK a value of 4.9 and a collective binding constant for zinc of 5 ؋ 10 ؊12 M at pH 7.0. The zinc cluster in the C-terminal ␣-domain is more stable than that in the ␤-domain. Its pH titration is also more complex, indicating at least two classes of zinc sites with different affinities. The whole molecule is stabilized with regard to the individual domains. Chemical modification implicates lysine side chains in both the stabilization of the ␤-domain cluster and the mutual stabilization of the domains in the whole molecule. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor molecule dependent on its type and characteristics. The isolated ␤-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated ␣-domain cluster, whereas the reverse is observed when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole molecule is important for its interaction with ligands and for control of its reactivity and overall conformation.