Metallomics最新文献

Metallothionein proteins are essential for Cu(I) and Zn(II) homeostasis as well as heavy metal detoxification. The metallation properties of MT2 are of great interest due to their wide patterns of expression and correlation with multiple diseases including cancers, neurological disorders, and respiratory diseases. Use of isotopically pure 63Cu(I) and 68Zn(II) eliminates the complexity of the Cu, Zn-MT2 mass spectral peaks due to significant overlap of naturally abundant isotopes. This allows for the resolution of the precise Cu(I) and Zn(II) stoichiometries when both Cu(I) and Zn(II) are bound to MT2 at physiological pH as expected in vivo. Exact Cu: Zn ratios were determined from mass spectral simulations carried out for every point in the titration. We report that Cu(I) metallation of Zn7-MT2 can only be understood in terms of two pathways occurring in parallel with pathway ① resulting in Cu5Zn5-MT2 and Cu9Zn3-MT2. Pathway ② results in Cu6Zn4-MT2 and Cu10Zn2-MT2, which are the major products of the reaction. From the electrospray ionization (ESI)-mass spectral data we report a series of formation constants (KF) for species starting from Zn7-MT2 up to Cu11Zn2-MT2. Room temperature phosphorescence and circular dichroism (CD) spectra were measured in parallel with the ESI-mass spectrometry data allowing for the assignment of specific species to specific spectral bands. Through analysis of the CD spectral bands, we propose that Cu(I) binds to the β domain first to form a Cu5Zn1 cluster or Cu6 cluster with emission at 670 and 750 nm, respectively, leaving the Zn4 cluster in the α domain.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare concern with associated healthcare costs reaching over ${$}$1 billion in a single year in the USA. Antibiotic resistance in S. aureus is now observed against last line of defense antibiotics, such as vancomycin, linezolid, and daptomycin. Unfortunately, high throughput drug discovery approaches to identify new antibiotics effective against MRSA have not resulted in much tangible success over the last decades. Previously, we demonstrated the feasibility of an alternative drug discovery approach, the identification of metallo-antibiotics, compounds that gain antibacterial activity only after binding to a transition metal ion and as such are unlikely to be detected in standard drug screens. We now report that avobenzone, the primary active ingredient of most sunscreens, can be activated by zinc to become a potent antibacterial compound against MRSA. Zinc-activated avobenzone (AVB-Zn) potently inhibited a series of clinical MRSA isolates [minimal inhibitory concentration (MIC): 0.62-2.5 µM], without pre-existing resistance and activity without zinc (MIC: >10 µM). AVB-Zn was also active against clinical MRSA isolates that were resistant against the commonly used zinc-salt antibiotic bacitracin. We found AVB-Zn exerted no cytotoxicity on human cell lines and primary cells. Last, we demonstrate AVB-Zn can be deployed therapeutically as lotion preparations, which showed efficacy in a mouse wound model of MRSA infection. AVB-Zn thus demonstrates Zn-activated metallo-antibiotics are a promising avenue for future drug discovery.

Synchrotron-based micro-X-ray fluorescence analysis (µXRF) is a nondestructive and highly sensitive technique. However, element mapping of rare earth elements (REEs) under standard conditions requires care, since energy-dispersive detectors are not able to differentiate accurately between REEs L-shell X-ray emission lines overlapping with K-shell X-ray emission lines of common transition elements of high concentrations. We aim to test REE element mapping with high-energy interference-free excitation of the REE K-lines on hyperaccumulator plant tissues and compare with measurements with REE L-shell excitation at the microprobe experiment of beamline P06 (PETRA III, DESY). A combination of compound refractive lens optics (CRLs) was used to obtain a micrometer-sized focused incident beam with an energy of 44 keV and an extra-thick silicon drift detector optimized for high-energy X-ray detection to detect the K-lines of yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), and neodymium (Nd) without any interferences due to line overlaps. High-energy excitation from La to Nd in the hyperaccumulator organs was successful but compared to L-line excitation less efficient and therefore slow (∼10-fold slower than similar maps at lower incident energy) due to lower flux and detection efficiency. However, REE K-lines do not suffer significantly from self-absorption, which makes XRF tomography of millimeter-sized frozen-hydrated plant samples possible. The K-line excitation of REEs at the P06 CRL setup has scope for application in samples that are particularly prone to REE interfering elements, such as soil samples with high concomitant Ti, Cr, Fe, Mn, and Ni concentrations.

Intracellular accumulation studies are a key step in metallodrug development but often variable results are obtained. Therefore, we aimed here to investigate different protocols for efficient and reproducible lysis of cancer cells in terms of protein content in lysates and in cell uptake studies of the Ru anticancer complex [chlorido(8-oxyquinolinato)(η6-p-cymene)ruthenium(II)] ([Ru(cym)(HQ)Cl]). The physical lysis methods osmosis and sonication were chosen for comparison with chemical lysis with the radioimmunoprecipitation assay (RIPA) buffer. Based on the protein content and the total Ru accumulated in the lysates, the latter determined using inductively coupled plasma-mass spectrometry, RIPA buffer was the most efficient lysis method. Measurements of plastic adsorption blanks revealed that the higher Ru content determined in the RIPA buffer lysis samples may be due a higher amount of Ru extracted from the plastic incubation plates compared with osmosis and sonication. Overall, we found that the choice of lysis method needs to be matched to the information sought and we suggest the least disruptive osmosis method might be the best choice for labile drug-biomolecule adducts. Minimal differences were found for experiments aimed at measuring the overall cell uptake of the Ru complex.

Thiosemicarbazones (TSCs) are a class of biologically active compounds with promising anticancer activity. Their typical mechanism, especially of the clinically far developed representative Triapine, is chelation of iron (Fe), with the Fe-containing enzyme ribonucleotide reductase as primary intracellular target. However, for the subclass of terminally disubstituted, nanomolar-active derivatives like Dp44mT and Me2NNMe2, recent findings suggest that the chelation, stability, and reduction properties of the copper(II) (Cu) complexes are essential for their modes of action. Consequently, it is important to elucidate whether blood serum Cu(II) is a potential metal source for these TSCs. To gain more insights, the interaction of Triapine, Dp44mT or Me2NNMe2 with purified human serum albumin (HSA) as the main pool of labile Cu(II) was investigated by UV-vis and electron paramagnetic resonance measurements. Subsequently, a size-exclusion chromatography inductively coupled plasma mass spectrometry method for the differentiation of Cu species in serum was developed, especially separating the non-labile Cu enzyme ceruloplasmin from HSA. The results indicate that the TSCs specifically chelate copper from the N-terminal Cu-binding site of HSA. Furthermore, the Cu(II)-TSC complexes were shown to form ternary HSA conjugates, most likely via histidine. Noteworthy, Fe-chelation from transferrin was not overserved, even not for Triapine. In summary, the labile Cu pool of HSA is a potential source for Cu-TSC complex formation and, consequently, distinctly influences the anticancer activity and pharmacological behavior of TSCs.

Pityrogramma calomelanos and Pteris vittata are cosmopolitan fern species that are the strongest known arsenic (As) hyperaccumulators, with potential to be used in the remediation of arsenic-contaminated mine tailings. However, it is currently unknown what chemical processes lead to uptake of As in the roots. This information is critical to identify As-contaminated soils that can be phytoremediated, or to improve the phytoremediation process. Therefore, this study identified the in situ distribution of As in the root interface leading to uptake in P. calomelanos and P. vittata, using a combination of synchrotron micro-X-ray fluorescence spectroscopy and X-ray absorption near-edge structure imaging to reveal chemical transformations of arsenic in the rhizosphere-root interface of these ferns. The dominant form of As in soils was As(V), even in As(III)-dosed soils, and the major form in P. calomelanos roots was As(III), while it was As(V) in P. vittata roots. Arsenic was cycled from roots growing in As-rich soil to roots growing in control soil. This study combined novel analytical approaches to elucidate the As cycling in the rhizosphere and roots enabling insights for further application in phytotechnologies to remediated As-polluted soils.

Epidemiological and animal studies have supported the carcinogenicity of hexavalent chromium [Cr(VI)]; however, molecular changes responsible for the induction of cancer by Cr(VI) are not entirely understood. Numerous mechanistic studies suggested the role of oxidative stress and genotoxicity in Cr(VI)-mediated carcinogenesis; however, specific types of DNA damage have not yet been conclusively attributed to specific chromium species or other reactive byproducts generated in biological systems exposed to Cr(VI). Due to the remarkably complex chemistry and biological effects of chromium species generated through the intracellular reduction of Cr(VI), their relevance for Cr(VI)-mediated carcinogenesis has not yet been fully elucidated and continues to be a subject of ongoing discussions in the field. In this report, we describe a complex world of chromium species and their reactivity with DNA and other biologically relevant molecules in vitro to inform a more complete understanding of Cr(VI)-mediated toxicity. In addition, we discuss previous results in the context of in vitro models and analytical methods to reconcile some conflicting findings on the biological role of chromium species.

As the second most abundant transition element and a crucial cofactor for many proteins, zinc is essential for the survival of all living organisms. To maintain required zinc levels and prevent toxic overload, cells and organisms have a collection of metal transport proteins for uptake and efflux of zinc. In bacteria, metal transport proteins are well defined for model organisms and many pathogens, but fewer studies have explored metal transport proteins, including those for zinc, in commensal bacteria from the gut microbiota. The healthy human gut microbiota comprises hundreds of species and among these, bacteria from the Lactobacillaceae family are well documented to have various beneficial effects on health. Furthermore, changes in dietary metal intake, such as for zinc and iron, are frequently correlated with changes in abundance of Lactobacillaceae. Few studies have explored zinc requirements and zinc homeostasis mechanisms in Lactobacillaceae, however. Here we applied a bioinformatics approach to identify and compare predicted zinc uptake and efflux proteins in several Lactobacillaceae genera of intestinal relevance. Few Lactobacillaceae had zinc transporters currently annotated in proteomes retrieved from the UniProt database, but protein sequence-based homology searches revealed that high-affinity ABC transporter genes are likely common, albeit with genus-specific domain features. P-type ATPase transporters are probably also common and some Lactobacillaceae genera code for predicted zinc efflux cation diffusion facilitators. This analysis confirms that Lactobacillaceae harbor genes for various zinc transporter homologs, and provides a foundation for systematic experimental studies to elucidate zinc homeostasis mechanisms in these bacteria.

The movement of metals through the environment links together a wide range of scientific fields: from earth sciences and geology as weathering releases minerals; to environmental sciences as metals are mobilized and transformed, cycling through soil and water; to biology as living things take up metals from their surroundings. Studies of these fundamental processes all require quantitative analysis of metal concentrations, locations, and chemical states. Synchrotron X-ray tools can address these requirements with high sensitivity, high spatial resolution, and minimal sample preparation. This perspective describes the state of fundamental scientific questions in the lifecycle of metals, from rocks to ecosystems, from soils to plants, and from environment to animals. Key X-ray capabilities and facility infrastructure for future synchrotron-based analytical resources serving these areas are summarized, and potential opportunities for future experiments are explored.

Despite their similar physicochemical properties, recent studies have demonstrated that lanthanides can display different biological behaviors. Hence, the lanthanide series can be divided into three parts, namely early, mid, and late lanthanides, based on their interactions with biological systems. In particular, the late lanthanides demonstrate distinct, but poorly understood biological activity. In the current study, we employed genome-wide functional screening to help understand biological effects of exposure to Yb(III) and Lu(III), which were selected as representatives of the late lanthanides. As a model organism, we used Saccharomyces cerevisiae, since it shares many biological functions with humans. Analysis of the functional screening results indicated toxicity of late lanthanides is consistent with disruption of vesicle-mediated transport, and further supported a role for calcium transport processes and mitophagy in mitigating toxicity. Unexpectedly, our analysis suggested that late lanthanides target proteins with SH3 domains, which may underlie the observed toxicity. This study provides fundamental insights into the unique biological chemistry of late lanthanides, which may help devise new avenues toward the development of decorporation strategies and bio-inspired separation processes.