Ratiometric fluorescent sensor proteins with subnanomolar affinity for Zn(II) based on copper chaperone domains

The ability to image the concentration of transition metals in living cells in real time is important for further understanding of transition metal homeostasis and its involvement in diseases. The goal of this study was to develop a genetically encoded FRET-based sensor for copper(I) based on the copper-induced dimerization of two copper binding domains involved in human copper homeostasis, Atox1 and the fourth domain of ATP7B (WD4). A sensor has been constructed by linking these copper binding domains to donor and acceptor fluorescent protein domains. Energy transfer is observed in the presence of Cu(I), but the Cu(I)-bridged complex is easily disrupted by low molecular weight thiols such as DTT and glutathione. To our surprise, energy transfer is also observed in the presence of very low concentrations of Zn(II) (10(-)(10) M), even in the presence of DTT. Zn(II) is able to form a stable complex by binding to the cysteines present in the conserved MXCXXC motif of the two copper binding domains. Co(II), Cd(II), and Pb(II) also induce an increase in FRET, but other, physiologically relevant metals are not able to mediate an interaction. The Zn(II) binding properties have been tuned by mutation of the copper-binding motif to the zinc-binding consensus sequence MDCXXC found in the zinc transporter ZntA. The present system allows the molecular mechanism of copper and zinc homeostasis to be studied under carefully controlled conditions in solution. It also provides an attractive platform for the further development of genetically encoded FRET-based sensors for Zn(II) and other transition metal ions.     

Translocation of Platinum Anticancer Drugs by Human Copper ATPases ATP7A and ATP7B

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down‐regulation of CTR1 and up‐regulation of the Cu‐ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu‐ATPases, we performed electrical measurements on the COS‐1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu‐ATPases and undergo ATP‐dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI‐MS to determine the binding mode of these drugs to the first N‐terminal metal‐binding domain of ATP7A (Mnk1).     

Structure and dynamics of Cu(I) binding in copper chaperones Atox1 and CopZ: a computer simulation study

Copper chaperones deliver reduced copper (i.e., Cu(I)) to metal-binding domains of P-type ATPases in the cytoplasm of a range of organisms. Both chaperones and target domains have a ferredoxin-like fold and metal-binding motifs involving two Cys residues. Here, we investigated the Cu-binding geometry and structural dynamics of two homologous Cu(I) chaperones, Homo sapiens Atox1 and Bacillus subtilis CopZ, using a combination of quantum mechanical-molecular mechanics (QM-MM) and classical molecular dynamics (MD) methods. Our QM-MM optimized geometries for the holo- proteins suggested that Cu(I) in Atox1 favors a linear Cys(S)-Cu-Cys(S) arrangement but that this angle is close to 150 degrees in CopZ. Classical MD simulations suggest that both Atox1 and CopZ apo- forms have an increased conformational flexibility as compared to the respective holo- forms. This difference is most pronounced in CopZ and correlates with a lower in vitro thermal stability. Both average fluctuation (i.e., rmsd) and radius of gyration data demonstrate that the effects of Cu(I) coordination extend throughout the proteins. Distinct deviations between the two homologues were found in protein-solvent interactions, entropy of Cu(I) binding, and apo-protein Cys-Cys distance distributions. Our in silico results provide new insights into copper chaperone behavior with direct implications for copper transport mechanisms in vivo.     

In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions

Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions.     

Structural Biology of Cisplatin Complexes with Cellular Targets: The Adduct with Human Copper Chaperone Atox1 in Aqueous Solution

Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution—the Pt atom binds to Cys12 and Cys15 while retaining the two ammine groups—the structural determinants of the adduct are not known. Here a structural model by hybrid Car–Parrinello density functional theory‐based QM/MM simulations is provided. The platinated site minimally modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experimental data. Our in silico/in vitro approach provides, together with previous studies, an unprecedented view into the structural biology of cisplatin–protein adducts.     

Designed to dissolve: suppression of colloidal aggregation of Cu(I)-selective fluorescent probes in aqueous buffer and in-gel detection of a metallochaperone

Due to the lipophilicity of the metal-ion receptor, previously reported Cu(I)-selective fluorescent probes form colloidal aggregates, as revealed by dynamic light scattering. To address this problem, we have developed a hydrophilic triarylpyrazoline-based fluorescent probe, CTAP-2, that dissolves directly in water and shows a rapid, reversible, and highly selective 65-fold fluorescence turn-on response to Cu(I) in aqueous solution. CTAP-2 proved to be sufficiently sensitive for direct in-gel detection of Cu(I) bound to the metallochaperone Atox1, demonstrating the potential for cation-selective fluorescent probes to serve as tools in metalloproteomics for identifying proteins with readily accessible metal-binding sites.     

Azole-bridged diplatinum anticancer compounds. Modulating DNA flexibility to escape repair mechanism and avoid cross resistance

Dinuclear azole-bridged Pt compounds bind to DNA helices, forming intrastrand crosslinks between adjacent guanines in a similar way to cisplatin. Their cytotoxic profile is, however, different from that of first and second generation Pt drugs in that they lack cross resistance in cisplatin-resistant cell lines. In contrast to cisplatin, which induces a large kink in DNA duplex, structural NMR studies and molecular dynamics simulations have shown that azole-bridged diplatinum compounds induce only small structural changes in double-stranded DNA. These structural differences have been invoked to explain the different cytotoxic profile of these compounds. Here, we show that in addition to the small structural changes in DNA, dinuclear Pt compounds also affect DNA minor groove flexibility in a different way than cisplatin. Free-energy calculations on azole-bridged diplatinum DNA adducts reveal that opening of the minor groove requires a higher free-energy cost (DeltaG ~ 7-15 kcal/mol) than in the corresponding cisplatin-DNA adduct (DeltaG ~ 0 kcal/mol). This could prevent minor groove binding proteins from binding to diplatinum-DNA adducts thus leading to a different cellular response than cisplatin and possibly decreasing the activity of excision repair enzymes. Although the development of drug resistance is a highly complex mechanism, our findings provide an additional rationale for the improved cytotoxic activity of these compounds in cell lines resistant to cisplatin.     

Lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper-translocating ATPase (ATP7A), but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1 (15)N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased toward 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met, whereas at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.     

A pairwise chemical genetic screen identifies new inhibitors of glucose transport

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.     

Targeted single-wall carbon nanotube-mediated Pt(IV) prodrug delivery using folate as a homing device

Most low-molecular-weight platinum anticancer drugs have short blood circulation times that are reflected in their reduced tumor uptake and intracellular DNA binding. A platinum(IV) complex of the formula c, c, t-[Pt(NH 3) 2Cl 2(O 2CCH 2CH 2CO 2H)(O 2CCH 2CH 2CONH-PEG-FA)] ( 1), containing a folate derivative (FA) at an axial position, was prepared and characterized. Folic acid offers a means of targeting human cells that highly overexpress the folate receptor (FR). Compound 1 was attached to the surface of an amine-functionalized single-walled carbon nanotube (SWNT-PL-PEG-NH 2) through multiple amide linkages to use the SWNTs as a "longboat delivery system" for the platinum warhead, carrying it to the tumor cell and releasing cisplatin upon intracellular reduction of Pt(IV) to Pt(II). The ability of SWNT tethered 1 to destroy selectively FR(+) vs FR(-) cells demonstrated its ability to target tumor cells that overexpress the FR on their surface. That the SWNTs deliver the folate-bearing Pt(IV) cargos into FR(+) cancer cells by endocytosis was demonstrated by the localization of fluorophore-labeled SWNTs using fluorescence microscopy. Once inside the cell, cisplatin, formed upon reductive release from the longboat oars, enters the nucleus and reacts with its target nuclear DNA, as determined by platinum atomic absorption spectroscopy of cell extracts. Formation of the major cisplatin 1,2-intrastrand d(GpG) cross-links on the nuclear DNA was demonstrated by use of a monoclonal antibody specific for this adduct. The SWNT-tethered compound 1 is the first construct in which both the targeting and delivery moieties have been incorporated into the same molecule; it is also the first demonstration that intracellular reduction of a Pt(IV) prodrug leads to the cis-{Pt((NH 3) 2} 1,2-intrastrand d(GpG) cross-link in nuclear DNA.     

Biological evaluation of redox stable cisplatin/Cu(II)-DNA adducts as potential anticancer agents

A series of non-enolizable β-diketonate-based copper(II) complexes with LCuCl₂ [L = Knoevenagel condensates of curcumin (Salcimine) and methylacetoacetate (SalMaA)-based Schiff bases] chromospheres as functional models of chemotherapy drug cisplatin were investigated for their covalent interaction with herring sperm DNA. The synthesis and structural characterization of 1a and 1b have been reported in our previous article. However, their DNA interactions and cytotoxicity properties were not studied. These analyses have been carried out mainly through electrochemical techniques supplemented with spectral, relative viscosity, gel electrophoresis techniques, and AGS cancer cells using MTT assay. The cytotoxic activities of the ligand, curcumin-based copper complex, and cisplatin were tested against the AGS cancer cell line under similar experimental conditions showing that the complex exhibited cancer cell inhibitory rate closer to cisplatin even at low concentration. This was also seen in the docking of the Cu-complex onto a rich guanine B-DNA decamer, where a Cu–N3_(guanine) interaction instead of Pt-N7 as cisplatin is detected. The obtained results in this study prove that these complexes could be a promising substitute for cisplatin as a new family of non-platinum-based anticancer metallo-drugs after in vivo tests on animal models.     

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.     

SITES OF PHOTOSENSITIZATION BY DERIVATIVES OF HEMATOPORPHYRIN

Leukemia L1210 cells were incubated in vitro with the tumor-localizing product HPD (hem-atoporphyrin derivative) for 0.5. 4 and 18 h. Effects of subsequent irradiation on viability, membrane transport and integrity, DNA synthesis and intracellular ATP concentration were assessed. Intracellular porphyrin pools were analyzed by HPLC. A 30 min incubation led to concentration of a readily-exchangeable pool of monomeric HPD components at plasma membrane loci; irradiation resulted in photodamage to membrane transport and a loss in capacity for dye exclusion. In contrast, increasing the incubation time led to a corresponding increase in the size of a non-exchangeable intracellular pool of other HPD components. Subsequent irradiation led to depletion of intracellular ATP and loss of capacity for biosynthesis of DNA, but little plasma membrane damage.     

Copper-binding properties and structures of methanobactins from Methylosinus trichosporium OB3b

Methanobactins (mbs) are a class of copper-binding peptides produced by aerobic methane oxidizing bacteria (methanotrophs) that have been linked to the substantial copper needs of these environmentally important microorganisms. The only characterized mbs are those from Methylosinus trichosporium OB3b and Methylocystis strain SB2. M. trichosporium OB3b produces a second mb (mb-Met), which is missing the C-terminal Met residue from the full-length form (FL-mb). The as-isolated copper-loaded mbs bind Cu(I). The absence of the Met has little influence on the structure of the Cu(I) site, and both molecules mediate switchover from the soluble iron methane mono-oxygenase to the particulate copper-containing enzyme in M. trichosporium OB3b cells. Cu(II) is reduced in the presence of the mbs under our experimental conditions, and the disulfide plays no role in this process. The Cu(I) affinities of these molecules are extremely high with values of (6-7) × 10(20) M(-1) determined at pH ≥ 8.0. The affinity for Cu(I) is 1 order of magnitude lower at pH 6.0. The reduction potentials of copper-loaded FL-mb and mb-Met are 640 and 590 mV respectively, highlighting the strong preference for Cu(I) and indicating different Cu(II) affinities for the two forms. Cleavage of the disulfide bridge results in a decrease in the Cu(I) affinity to ～9 × 10(18) M(-1) at pH 7.5. The two thiolates can also bind Cu(I), albeit with much lower affinity (～ 3 × 10(15) M(-1) at pH 7.5). The high affinity of mbs for Cu(I) is consistent with a physiological role in copper uptake and protection.     

Fluorescence assay for monitoring Zn-deficient superoxide dismutase in vitro

A method has been developed for selective detection of the zinc-deficient form of Cu, Zn superoxide dismutase (SOD1) in vitro. Zinc-deficient SOD1 mutants have been implicated in the death of motor neurons leading in amyotrophic lateral sclerosis (ALS or Lou Gerhig's disease). Thus, this method may have applicability for detecting zinc-deficient SOD1 mutants in human ALS patients samples as well as in a transgenic mouse model of ALS and in cultured motor neurons. We determined previously that structural analogs of 1,10 phenanthroline, which react specifically with Cu(I), react with the active Cu(I) of SOD1 when zinc is absent, but not when zinc is also bound, as evidenced by the fact that the reaction is inhibited by pretreatment of the enzyme with zinc. We report herein that bathocuproine, or its water-soluble derivative bathocuproine disulfonate, react with zinc-deficient SOD1 to form a complex which fluoresces at 734 nm when excited at 482 nm. Fluorescent intensity is concentration dependent, thus we propose to use fluorescent confocal microscopy to measure intracellular levels of zinc-deficient SOD1 in situ.     

Insights into the N-terminal Cu(II) and Cu(I) binding sites of the human copper transporter CTR1

Abstract

Copper transporter 1 (CTR1) is the main copper transporter in the eukaryotic system. CTR1 has several important roles: It binds Cu(II) ions that are present in the blood; it reduces those Cu(II) ions to Cu(I); and it subsequently transfers Cu(I) to the cytoplasmic domain, where the ion is delivered to various cellular pathways. Here, we seek to identify CTR1 binding sites for Cu(II) and Cu(I) and to shed light on the Cu(II)-to-Cu(I) reduction process. We focus on the first 14 amino acids of CTR1. This N-terminal segment is rich with histidine and methionine residues, which are known to bind Cu(II) and Cu(I), respectively; thus, this region has been suggested to have an important function in recruiting Cu(II) and reducing it to Cu(I). We utilize electron paramagnetic resonance (EPR) spectroscopy together with nuclear magnetic resonance (NMR) and UV-VIS spectroscopy and alanine substitution to reveal Cu(II) and Cu(I) binding sites in the focal 14-amino-acid segment. We show that H5 and H6 directly coordinate to Cu(II), whereas M7, M9, and M12 are involved in Cu(I) binding. This research is another step on the way to a complete understanding of the cellular copper regulation mechanism in humans.     

Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin‐binding proteins. The method used a fluorescent carboxyfluorescein‐diacetate‐labeled cisplatin analogue (CFDA‐cisplatin), two‐dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA‐cisplatin‐containing protein adducts in the acidic pH range (pH 4–7). Based on this analytical method we extended the identification of intracellular cisplatin‐protein adducts to the alkaline pH range (pH 6–10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA‐cisplatin‐binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D‐3‐phosphoglycerate 1‐dehydrogenase.