Zhaoyong Xi

Copper binding promotes the interaction of cisplatin with human copper chaperone Atox1

Cu(I) binding promotes the platination of Atox1, although cisplatin binds to the copper coordination sites. In addition, Cu(I) binding enhances the competition of Atox1 with DTT in the reaction of cisplatin. These results indicate that cuprous ions could regulate the cellular trafficking of cisplatin.

Site‐specific 19F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid

Site‐specific 19F chemical shift and side chain relaxation analysis can be applied on large size proteins. Here, one‐dimensional 19F spectra and T1, T2 relaxation data were acquired on a SH3 domain in aqueous buffer containing 60% glycerol, and a nine‐transmembrane helices membrane protein diacyl‐glycerol kinase (DAGK) in dodecyl phosphochoine (DPC) micelles. The high quality of the data indicates that this method can be applied to site‐specifically analyze side chain internal mobility of membrane proteins or large size proteins.

Trans‐Platinum/Thiazole Complex Interferes with Sp1 Zinc‐Finger Protein

Abstract No trafficking: The antitumor-active trans-platinum/thiazole complex trans-PtTz demonstrated high reactivity to the transcription factor Sp1, which is overexpressed in tumor cells. The binding of trans-PtTz disrupts the DNA interaction with Sp1 in vitro and prevents the protein trafficking from the cytoplasm into the nucleus.

Cisplatin binds to human copper chaperone Cox17: the mechanistic implication of drug delivery to mitochondria

Cox17 facilitates the platinum accumulation in mitochondria, which contributes to the overall cytotoxicity of cisplatin.

Transporting platinum drugs from a copper chaperone to ATPase: the mechanistic implication of drug efflux mediated cisplatin resistance

Cisplatin transfers from Atox1 to ATPase, which could be associated with drug efflux from the cell.

The Reaction of Arsenite with Proteins Relies on Solution Conditions

Arsenic is a biologically interesting element with both antitumor and carcinogenic effects. Zinc finger proteins (ZFPs) have been confirmed to be the cellular targets of arsenite; however, arsenite inhibits ZFPs much less efficiently in vitro than in vivo. The molecular mechanism of this difference is unknown. In this work, we found that the reaction of arsenite with ZFPs relies on the presence of small biomolecules such as glutathione (GSH), histidine, and cysteine (Cys). The weak acidity also enhances the reaction. Further study shows that the coordination of zinc is much more susceptible than that of arsenic to these solution conditions, which enhance the competition of arsenic. Notably, different from C3H-type ZFPs, the C2H2-type ZFPs are more significantly influenced by the presence of thiol-containing molecules in the reaction. GSH and Cys can facilitate the reaction by participation of the coordination to As(III) together with C2H2-type ZFPs. Consequently, the reactions are promoted both thermodynamically and kinetically via the formation of ternary complexes GSH-As-ZFP or Cys-As-ZFP. These results indicate that the reactions between arsenite and proteins are considerably modulated by environments such as the small biomolecules and the acidity of the solution. This finding clarifies the discrepancy observed in the reactions of arsenite in vitro versus in cells, and provides an insight into the molecular mechanism of arsenite.

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded 15N/19F-labeled unnatural amino acid.

SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for (15)N/(19)F-trifluoromethyl-phenylalanine ((15)N/(19)F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ((15)N)(1)H and side-chain (19)F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ((15)N)(1)H and side-chain (19)F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

Copper binding modulates the platination of human copper chaperone Atox1 by antitumor trans-platinum complexes

The transport system of platinum-based anticancer agents is crucial for drug sensitivity. Increasing evidence indicates that the copper transport system is also involved in the cellular influx and efflux of platinum drugs. The copper chaperone Atox1 has been shown to bind to cisplatin in vitro and in cells. Previous results reveal that copper binding promotes the reaction between Atox1 and cisplatin. Here, we have performed detailed solution NMR and ESI-MS experiments to investigate the effect of Cu(i) binding on the reactions of Atox1 with two antitumor active trans-platinum agents, trans-EE and trans-PtTz. Results indicate that, similar to the reaction of cisplatin, copper coordination also enhances the platination of Atox1 by two trans-platinum complexes, and platinum binds to the copper coordinating residues. However, copper binding promotes the trans-platinum transfer from Atox1 to dithiothreitol (DTT). This result is in contrast to the reaction of Atox1 with cisplatin, in which the presence of copper largely suppresses the platination of DTT. Additionally, both apo- and Cu(I)-Atox1 react faster with trans-platinum complexes than with cisplatin, however, less protein aggregation is observed in the reaction of trans-platinum complexes. These results indicate that the roles of Atox1 in the regulation of cellular trafficking of platinum drugs are dependent on the coordination configurations.

Glutathione selectively modulates the binding of platinum drugs to human copper chaperone Cox17

The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem. Commun. 50: , 2667-2669]. Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment.

Conserved residue modulates copper-binding properties through structural dynamics in human copper chaperone Atox1

The human copper chaperone Atox1 plays a central role in the transport of copper in cells. It has been reported that the conserved residue Lys60 contributes to the heterocomplex stability of Atox1 with its target protein ATPase, and that the K60A mutation could diminish the copper transfer. In this work, we carried out the structure determination and dynamic analysis of Atox1 with the K60A mutation in order to elucidate the role of the conserved residue Lys60 in the copper transport. Results show that the K60A mutation results in crucial secondary structure rearrangements and side-chain orientation alteration of the metal-binding residues in Atox1. Protein dynamic studies reveal that the K60A mutation leads to increased overall flexibility, and a significant difference in dynamic properties of the metal-binding sites. The structure and dynamic changes cause a decrease in the copper-binding stability of the K60A mutant. In addition, Cu(i)-mediated hetero-protein interactions with ATP7A are present in the metal transfer of both Atox1 variants, although copper transfer is accompanied with smaller structural alteration in the K60A mutant. These results indicate that Lys60 is crucial in maintaining the structure and dynamic properties of Atox1.