Combinations of arginine and pullulan reveal the selective effect of stabilization mechanisms on different lyophilized proteins.

The stability of lactate dehydrogenase (LDH) and ß-galactosidase (ß-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while ß-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, ß-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and ß-gal may be dependent on the interaction with arginine.

Photocontrol of the ß-Hairpin Polypeptide Structure through an Optimized Azobenzene-Based Amino Acid Analogue.

A family of neurodegenerative diseases, including Huntington's disease (HD) and spinocerebellar ataxias, are associated with an abnormal polyglutamine (polyQ) expansion in mutant proteins that become prone to form amyloid-like aggregates. Prior studies have suggested a key role for ß-hairpin formation as a driver of nucleation and aggregation, but direct experimental studies have been challenging. Toward such research, we set out to enable spatiotemporal control over ß-hairpin formation by the introduction of a photosensitive ß-turn mimic in the polypeptide backbone, consisting of a newly designed azobenzene derivative. The reported derivative overcomes the limitations of prior approaches associated with poor photochemical properties and imperfect structural compatibility with the desired ß-turn structure. A new azobenzene-based ß-turn mimic was designed, synthesized, and found to display improved photochemical properties, both prior and after incorporation into the backbone of a polyQ polypeptide. The two isomers of the azobenzene-polyQ peptide showed different aggregate structures of the polyQ peptide fibrils, as demonstrated by electron microscopy and solid-state NMR (ssNMR). Notably, only peptides in which the ß-turn structure was stabilized (azobenzene in the cis configuration) closely reproduced the spectral fingerprints of toxic, ß-hairpin-containing fibrils formed by mutant huntingtin protein fragments implicated in HD. These approaches and findings will enable better deciphering of the roles of ß-hairpin structures in protein aggregation processes in HD and other amyloid-related neurodegenerative diseases.

Magnetic resonance investigation of conformational responses of tau protein to specific phosphorylation.

Intrinsically disordered proteins (IDPs) are known to adopt many rapidly interconverting structures, making it difficult to pinpoint the specific conformational states that are relevant for their function. Tau is an important IDP, and its conformation is known to be affected by post-translational modifications (PTMs), such as phosphorylation. To investigate the effect of specific phosphorylation on full-length Tau's dynamic global conformation, we employed a combination of nuclear magnetic resonance-based paramagnetic relaxation interference methods and electron paramagnetic resonance spectroscopy. By reproducing the AT8 epitope, comprising exclusive phosphorylation at residues S202 and T205, we were able to identify conformations specific to phosphorylated Tau, which exhibited a tendency towards less compact states. These mechanistic details are of significance to understand the path leading from soluble Tau to the ordered structure of Tau fibers. This approach proved to be successful for studying the conformational changes of (phosphorylated) full-length Tau and can potentially be extended to the study of other IDPs that undergo various PTMs.

Anomalous peroxidase activity of cytochrome c is the primary pathogenic target in Barth syndrome.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL-cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL-cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL-cyt c peroxidase offers therapeutic approaches to BTHS treatment.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Conformation and Affinity Modulations by Multiple Phosphorylation Occurring in the BIN1 SH3 Domain Binding Site of the Tau Protein Proline-Rich Region.

An increase in phosphorylation of the Tau protein is associated with Alzheimer's disease (AD) progression through unclear molecular mechanisms. In general, phosphorylation modifies the interaction of intrinsically disordered proteins, such as Tau, with other proteins; however, elucidating the structural basis of this regulation mechanism remains challenging. The bridging integrator-1 gene is an AD genetic determinant whose gene product, BIN1, directly interacts with Tau. The proline-rich motif recognized within a Tau(210-240) peptide by the SH3 domain of BIN1 (BIN1 SH3) is defined as 216PTPP219, and this interaction is modulated by phosphorylation. Phosphorylation of T217 within the Tau(210-240) peptide led to a 6-fold reduction in the affinity, while single phosphorylation at either T212, T231, or S235 had no effect on the interaction. Nonetheless, combined phosphorylation of T231 and S235 led to a 3-fold reduction in the affinity, although these phosphorylations are not within the BIN1 SH3-bound region of the Tau peptide. Using nuclear magnetic resonance (NMR) spectroscopy, these phosphorylations were shown to affect the local secondary structure and dynamics of the Tau(210-240) peptide. Models of the (un)phosphorylated peptides were obtained from molecular dynamics (MD) simulation validated by experimental data and showed compaction of the phosphorylated peptide due to increased salt bridge formation. This dynamic folding might indirectly impact the BIN1 SH3 binding by a decreased accessibility of the binding site. Regulation of the binding might thus not only be due to local electrostatic or steric effects from phosphorylation but also to the modification of the conformational properties of Tau.

New insights in polydopamine formation via surface adsorption.

Polydopamine is a biomimetic self-adherent polymer, which can be easily deposited on a wide variety of materials. Despite the rapidly increasing interest in polydopamine-based coatings, the polymerization mechanism and the key intermediate species formed during the deposition process are still controversial. Herein, we report a systematic investigation of polydopamine formation on halloysite nanotubes; the negative charge and high surface area of halloysite nanotubes favour the capture of intermediates that are involved in polydopamine formation and decelerate the kinetics of the process, to unravel the various polymerization steps. Data from X-ray photoelectron and solid-state nuclear magnetic resonance spectroscopies demonstrate that in the initial stage of polydopamine deposition, oxidative coupling reaction of the dopaminechrome molecules is the main reaction pathway that leads to formation of polycatecholamine oligomers as an intermediate and the post cyclization of the linear oligomers occurs subsequently. Furthermore, TRIS molecules are incorporated into the initially formed oligomers.

Structural Dynamics and Tunability for Colloidal Tin Halide Perovskite Nanostructures.

Lead halide perovskite nanocrystals are highly attractive for next-generation optoelectronics because they are easy to synthesize and offer great compositional and morphological tunability. However, the replacement of lead by tin for sustainability reasons is hampered by the unstable nature of Sn2+ oxidation state and by an insufficient understanding of the chemical processes involved in the synthesis. Here, an optimized synthetic route is demonstrated to obtain stable, tunable, and monodisperse CsSnI3 nanocrystals, exhibiting well-defined excitonic peaks. Similar to lead halide perovskites, these nanocrystals are prepared by combining a precursor mixture of SnI2 , oleylamine, and oleic acid, with a Cs-oleate precursor. Among the products, nanocrystals with 10 nm lateral size in the γ-orthorhombic phase prove to be the most stable. To achieve such stability, an excess of precursor SnI2 as well as substoichiometric Sn:ligand ratios are key. Structural, compositional, and optical investigations complemented by first-principle density functional theory calculations confirm that nanocrystal nucleation and growth follow the formation of (R-NH3 + )2 SnI4 nanosheets, with R = C18 H35 . Under specific synthetic conditions, stable mixtures of 3D nanocrystals CsSnI3 and 2D nanosheets (Ruddlesden-Popper (R-NH3 + )2 Csn -1 Snn I3 n +1 with n > 1) are obtained. These results set a path to exploiting the high potential of Sn halide perovskite nanocrystals for opto-electronic applications.

Interaction of Copper Trafficking Proteins with the Platinum Anticancer Drug Kiteplatin.

The interaction of metallodrugs with proteins influences their mechanism of action and side effects. In the case of platinum drugs, copper transporters modulate sensitivity and resistance to these anticancer agents. To deepen the knowledge of the structural properties underlying the reactivity of platinum drugs with copper transporters, we studied the interaction of kiteplatin and two of its derivatives with the methionine-rich motif of copper importer Ctr1 and with the dithiol motif of the first domain of Menkes ATPase. Furthermore, cellular uptake and cytotoxicity of the three complexes were evaluated in cisplatin-sensitive and -resistant ovarian cancer cells, comparing the data with those of clinically relevant drugs. Reactivity depends on the tightness of the chelate ring formed by the carrier ligands and the nature of the leaving and entering groups. The results highlight the importance of subtle changes in the platinum coordination sphere that affect drug absorption and intracellular fate.

Protofilament Structure and Supramolecular Polymorphism of Aggregated Mutant Huntingtin Exon 1.

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.

Role of Tau as a Microtubule-Associated Protein: Structural and Functional Aspects.

Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer's disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.

BIN1 recovers tauopathy-induced long-term memory deficits in mice and interacts with Tau through Thr348 phosphorylation.

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.

Nuclear Magnetic Resonance Spectroscopy Insights into Tau Structure in Solution: Impact of Post-translational Modifications.

Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure. The cis or trans conformation of X-Proline peptide bonds is an additional characteristic parameter of Tau structure that is targeted and modified by prolyl cis/trans isomerases. The challenge in molecular characterization of Tau lies in being able to link structural parameters to functional consequences in normal functions and dysfunctions of Tau, including potential misfolding on the path to aggregation and/or perturbation of the interactions of Tau with its many molecular partners. Phosphorylation of Ser and Thr residues has the potential to impact the local and global structure of Tau.

Structural Basis of Tau Interaction With BIN1 and Regulation by Tau Phosphorylation.

Bridging integrator-1 (BIN1) gene is associated with an increased risk to develop Alzheimer's disease, a tauopathy characterized by intra-neuronal accumulation of phosphorylated Tau protein as paired helical filaments. Direct interaction of BIN1 and Tau proteins was demonstrated to be mediated through BIN1 SH3 C-terminal domain and Tau (210-240) peptide within Tau proline-rich domain. We previously showed that BIN1 SH3 interaction with Tau is decreased by phosphorylation within Tau proline-rich domain, of at least T231. In addition, the BIN1/Tau interaction is characterized by a dynamic equilibrium between a closed and open conformations of BIN1 isoform 1, involving an intramolecular interaction with its C-terminal BIN1 SH3 domain. However, the role of the BIN1/Tau interaction, and its potential dysregulation in Alzheimer's disease, is not yet fully understood. Here we showed that within Tau (210-240) peptide, among the two proline-rich motifs potentially recognized by SH3 domains, only motif P216TPPTR221 is bound by BIN1 SH3. A structural model of the complex between BIN1 SH3 and Tau peptide (213-229), based on nuclear magnetic resonance spectroscopy data, revealed the molecular detail of the interaction. P216 and P219 within the proline-rich motif were in direct contact with the aromatic F588 and W562 of the BIN1 SH3 domain. The contact surface is extended through electrostatic interactions between the positively charged R221 and K224 residues of Tau peptide and those negatively charged of BIN1 SH3, corresponding to E556 and E557. We next investigated the impact of multiple Tau phosphorylations within Tau (210-240) on its interaction with BIN1 isoform 1. Tau (210-240) phosphorylated at four different sites (T212, T217, T231, and S235), contrary to unphosphorylated Tau, was unable to compete with the intramolecular interaction of BIN1 SH3 domain with its CLAP domain. In accordance, the affinity of BIN1 SH3 for phosphorylated Tau (210-240) peptide was reduced, with a five-fold increase in the dissociation constant, from a Kd of 44 to 256 µM. This study highlights the complexity of the regulation of BIN1 isoform 1 with Tau. As abnormal phosphorylation of Tau is linked to the pathology development, this regulation by phosphorylation might have important functional consequences.

Monitoring Interactions Inside Cells by Advanced Spectroscopies: Overview of Copper Transporters and Cisplatin.

BACKGROUND: Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum- based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation. METHODS: The use of powerful spectroscopic, spectrometric and computational methods has allowed a deep structural insight into the mode of interaction of platinum drugs with the metal-binding domains of the transporter proteins. RESULTS: This review article focuses on the mode in which platinum drugs can compete with copper ion for binding to transport proteins and consequent structural and biological effects. Three types of transporters are discussed in detail: copper transporter 1 (Ctr1), the major responsible for Cu+ uptake; antioxidant-1 copper chaperone (Atox1), responsible for copper transfer within the cytoplasm; and copper ATPases (ATP7A/B), responsible for copper export into specific subcellular compartments and outside the cell. CONCLUSION: The body of knowledge summarized in this review can help in shaping current chemotherapy to optimize the efficacy of platinum drugs (particularly in relation to resistance) and to mitigate adverse effects on copper metabolism.

Activation of Platinum(IV) Prodrugs by Cytochrome c and Characterization of the Protein Binding Sites.

Platinum(IV) complexes generally require reduction to reactive Pt(II) species to exert their chemotherapeutic activity. The process of reductive activation of (15)N-labeled (OC-6-43)-bis(acetato)diamminedichloridoplatinum(IV), in the presence of nicotinamide adenine dinucleotide (NADH) and horse heart cytochrome c (cyt c), was monitored by (1)H,(15)N-HSQC NMR spectroscopy and protein digestion experiments. It has been shown that cyt c plays a catalytic role in the transfer of two reducing equivalents from NADH to Pt(IV) species. Noncovalent interactions between reduced monoaqua cisplatin (cis-[PtCl((15)NH3)2(H2O)](+)) and the protein, in the proximity of the heme cofactor, and also covalent binding of platinum to the protein region around Met65 and Met80 take place.

Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.

Cisplatin handover between copper transporters: the effect of reducing agents.

Copper (Cu) transporters emerged as key factors at the basis of the biological response to antitumor platinum (Pt) drugs, which are among the most potent and broadly used chemotherapeutics. ATP7A and ATP7B (the Menkes and Wilson disease proteins, respectively) appear to be implicated in promoting tumor cell resistance to cisplatin. Cu-ATPases could bind the drug and, with the alleged involvement of the chaperone ATOX1, contribute to cell detoxification and survival. Here, we report the spectroscopic characterization of cisplatin binding to ATOX1 and MNK1, the first metal-binding domain of ATP7A, in the presence of the physiological reducing agent glutathione, a sulfur-containing molecule responsible for the majority of Pt detoxification in the cytosol. Under conditions mimicking the cellular environment, we show that cisplatin transfer from ATOX1 to MNK1 does not occur at a detectable rate. These results appear to contradict other literature data which, however, were obtained in the presence of exogenous reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) having good coordinating ability for soft metal ions (such as Pt) and strong trans-labilizing effect. A better understanding of Pt drug processing by Cu trafficking proteins under physiological conditions may help to answer key issues, such as drug availability in tumor cells and resistance.