Investigation on the influence of (Z)-3-(2-(3-chlorophenyl)hydrazono)-5,6-dihydroxyindolin-2-one (PT2) on ß-amyloid(1-40) aggregation and toxicity.

In Alzheimer's disease (AD), native Aß protein monomers aggregate through the formation of a variety of water-soluble, toxic oligomers, ultimately leading to insoluble fibrillar deposits. The inhibition of oligomers formation and/or their dissociation into non-toxic monomers, are considered an attractive strategy for the prevention and treatment of AD. A number of studies have demonstrated that small molecules, containing single or multiple (hetero)aromatic rings, can inhibit protein aggregation, being potentially effective in AD treatment. Starting from previously reported data on the antiamyloidogenic activity of a series of 3-hydrazonoindolinones, compound PT2 was selected to deeply investigate the inhibitory mechanism in the Aß aggregation cascade. We compared data from DLS, NMR, CD, TEM and ThT fluorescence measures to ascertain the interactions with amyloidogenic species formed in vitro during the aggregation process, and confirmed this feature with cell viability tests on HeLa cultured cells. PT2 was effective in disrupting toxic oligomers and mature amyloid fibrils, stabilizing Aß as non-toxic, ß-sheet arranged, ThT-insensitive protofilaments. It also strongly reduced cellular toxicity caused by Aß and showed good antioxidant properties in two radical scavenging tests. Taken together, these data confirmed that PT2 is a small molecule inhibitor of Aß oligomerization and toxicity, displaying also additional activity as antioxidant.

Synthesis, Characterization, and in Vitro Evaluation of a New TSPO-Selective Bifunctional Chelate Ligand.

The 18-kDa translocator protein (TSPO) is overexpressed in many types of cancers and is also abundant in activated microglial cells occurring in inflammatory neurodegenerative diseases. Thus, TSPO has become an extremely attractive subcellular target not only for imaging disease states overexpressing this protein, but also for a selective mitochondrial drug delivery. In this work we report the synthesis, the characterization, and the in vitro evaluation of a new TSPO-selective ligand, 2-(8-(2-(bis(pyridin-2-yl)methyl)amino)acetamido)-2-(4-chlorophenyl)H-imidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide (CB256), which fulfils the requirements for a bifunctional chelate approach. The goal was to provide a new TSPO ligand that could be used further to prepare coordination complexes of a metallo drug to be used in diagnosis and therapy. However, the ligand itself proved to be a potent tumor cell growth inhibitor and DNA double-strand breaker.

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.

C60@Lysozyme: direct observation by nuclear magnetic resonance of a 1:1 fullerene protein adduct.

Integrating carbon nanoparticles (CNPs) with proteins to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science. The comprehension of CNP-protein interactions requires the still-missing identification and characterization of the 'binding pocket' for the CNPs. Here, using Lysozyme and C60 as model systems and NMR chemical shift perturbation analysis, a protein-CNP binding pocket is identified unambiguously in solution and the effect of the binding, at the level of the single amino acid, is characterized by a variety of experimental and computational approaches. Lysozyme forms a stoichiometric 1:1 adduct with C60 that is dispersed monomolecularly in water. Lysozyme maintains its tridimensional structure upon interaction with C60 and only a few identified residues are perturbed. The C60 recognition is highly specific and localized in a well-defined pocket.

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).

Probing the interaction of cisplatin with the human copper chaperone Atox1 by solution and in-cell NMR spectroscopy.

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH(3))(2)}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation.

Effect of thioethers on DNA platination by trans-platinum complexes.

Increasing evidence indicates that sulfur-containing molecules can play important roles in the activity of platinum anticancer drugs. Although nuclear DNA is retained to be the ultimate target, these platinum compounds can readily react with a variety of other substrates containing a soft donor atom, such as proteins, peptides, and low molecular weight biomolecules, before reaching DNA. In a recent study it was demonstrated that the DNA platination rate of a trans-geometry antitumor drug was dramatically enhanced by methionine binding, thus suggesting that the thioether could serve as a catalyst for DNA platination. In this work we performed detailed studies on the reactions of a widely investigated and very promising trans-platinum complex having two iminoethers and two chlorido ligands, trans-EE, with methionine (Met) and guanosine 5'-monophosphate (GMP). The results show that in the reaction of trans-EE with methionine the bisadduct is the dominant species in the early stage of the reaction. The reaction is also influenced by chloride concentration: at low NaCl the bis-methionine adduct is formed in preference, whereas the monoadduct is favored at high NaCl concentration. Not only the monomethionine complex, trans-PtCl(E-iminoether)(2)(AcMet), but also the bis-methionine adduct, trans-Pt(E-iminoether)(2)(AcMet)(2), which has already lost both leaving chlorides, can react with GMP to form the ternary platinum complex trans-Pt(E-iminoether)(2)(AcMet)(GMP). The latter reaction discloses the possibility of direct coordination to DNA of a platinum-protein adduct, in which the two carrier ligands remain intact; this is not the case of cis-oriented platinum complexes, like cisplatin, for which formation of a ternary complex is usually accompanied by loss of at least one carrier ligand. Interestingly, isomerization from S to N coordination of one methionine takes place in the bis-methionine complex at neutral pH, while the monoadduct appears to be stable. The shift from S to N coordination of one methionine in the trans-bis-methionine adduct can easily account for the obtainment of the cis isomer in the bis-chelated Pt(Met-S,N)(2) end product.

Copper-triggered aggregation of ubiquitin.

Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80:20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing beta-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates.

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies.

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.

Variable structures of promoters regulating transcription of cp-like tRNA genes and of some native genes on the sunflower mitochondrial genome.

Promoter regions regulating the transcription of all cp-like tRNA genes encoded by the sunflower chondriome have been identified. Some of these genes are part of clusters where the first gene is a typical mitochondrial isoform. Promoters regulating the transcription of single cp-like tRNA genes have a variable structure whereas those regulating the transcription of native genes or clusters with typical mitochondrial genes in the first position conform to a similar common structure. The variability of promoter regions described in this paper could be the result of modifications of regions having, at the moment of the cpDNA insertion event, only minimal structural features as promoters.