Metallomics最新文献

The gadolinium-based contrast agent Gadoxetic acid and the platinum-based antitumor agent Cisplatin were quantitatively imaged in liver and liver cancer (hepatocellular carcinoma, HCC) tissue of rats by means of laser ablation-inductively coupled plasma-mass spectrometry. HCC bearing rats simultaneously received a tail vein injection of the hepatocyte-specific magnetic resonance imaging contrast agent Gadoxetic acid and a transarterial injection of Cisplatin 15 min before sacrifice and liver removal. Resecting HCC with adjacent liver tissue allows the comparison of Gd, Pt, and endogenous elements like Fe, Cu, and Zn in the various tissue types. Region of interest analysis reveals lower concentrations of Gd in HCC and higher Gd content in the adjacent liver, fitting the selective uptake of Gadoxetic acid into hepatocytes. Furthermore, two malignancy grades and their possible impact on the Gadoxetic acid and Cisplatin uptake are compared. For this, four high grade (G3) and two moderate grade (G2) HCCs were analysed, including a control sample each. Gd concentrations were lower in HCC irrespective of the grade of dedifferentiation (G2, G3) compared to adjacent liver. Despite local arterial Cisplatin injection, concentrations of Pt were similar or also reduced in HCC compared to liver tissue. In addition, endogenous Fe, Cu, and Zn were quantified. While Zn was homogenously distributed, higher Fe concentrations were determined in liver tissue compared to HCC. Hotspots of Cu suggest a deregulated copper homeostasis in certain liver lesions. The Gd and Fe distributions are compared in detail with cellular alterations examined by hematoxylin and eosin staining.

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (μXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

Metallothioneins (MT) are regulators of the metals Zn(II) and Cu(I) and act as antioxidants in many organisms, including in humans. Isoform 3 (MT3) is expressed constitutively in central nervous tissue and has been shown to have additional biological functions, including the inhibition of neuronal growth, the regulation of apoptosis, and cytoskeleton modulation. To facilitate these functions, protein-protein interactions likely occur. These interactions may then impact the metalation status of the MT and the recipient metalloprotein. Using electrospray ionization mass spectrometry and circular dichroism spectroscopy, we report that the interaction between the zinc metalloenzyme, carbonic anhydrase (CA), and MT3, impacts the metalation profiles of both apo-MT3 and apo-CA with Cd(II) and Zn(II). We observe two phases in the metalation of the apo-CA, the first of which is associated with an increased binding affinity of apo-CA for Cd/Zn(II) and the second pathway is associated with apo-CA metalated without a change in binding affinity. The weak interactions that result in this change of binding affinity are not detectable as a protein complex in the ESI-mass spectral data or in the circular dichroism spectra. These unusual metalation properties of apo-CA in the presence of apo-MT3 are evidence of the effects of protein-protein interactions. With adjustment to take into account the interaction of both proteins, we report the complete Cd(II) and Zn(II) binding constants of MT3 under physiological conditions, as well as the pH dependence of these binding pathways.

Anemia of inflammation (or inflammation-associated anemia) decreases the quality of life in billions of patients suffering from various inflammatory diseases, such as infection, autoimmune diseases, and cancer, associated with a prolonged state of immune activation. While proper utilization of iron, a nutrient metal essential for erythropoiesis, is important for the prevention of anemia, the alteration of body iron homeostasis upon inflammation, which can contribute to the development of anemia, is not completely understood. Thus, we sought to examine temporal and spatial changes in the distribution of iron and iron-associated molecules during inflammation in mice. To induce inflammation, C57BL/6J mice were injected with turpentine oil weekly for 3 weeks, which resulted in anemia, decreased protein expression of ferroportin, a cellular iron exporter, in the spleen, duodenum, and liver, and increased iron stores in the duodenum and spleen. Tracer kinetic studies after oral administration of 59Fe revealed that more iron was found in the spleen and less in the femur bone in turpentine oil-injected mice compared to the saline-injected mice, indicating tissue-specific abnormalities in iron distribution during inflammation. However, there was no difference in the utilization of iron for red blood cell production after turpentine oil injection; instead, serum hemopexin level and lactate dehydrogenase activity were increased, suggesting increased red blood cell destruction upon inflammation. Our findings provide an improved understanding of temporal and spatial changes in the distribution and utilization of iron during inflammation.

Mutational inactivation of the P-type Cu-ATPase ATP7B interferes with its cellular functions to varying extent leading to varied cellular phenotypes. Wilson's disease (WD) primarily affects organs composed of polarized/differentiated epithelial cells. Therefore, phenotypic variability might differ depending on the polarization/differentiation of the cells. The present study investigates the intracellular stability and localization of ATP7B harboring WD mutations in both unpolarized/undifferentiated and polarized/differentiated cell-based models. Green fluorescent protein (GFP)-ATP7B harboring the WD causing mutations, N41S, S653Y, R778Q, G1061E, H1069Q, S1423N, S1426I, and T1434M, are included for investigation. The C-terminal WD mutations (S1423N, S1426I, and T1434M), exhibit distinct localization and Cu(I) responsive anterograde and retrograde trafficking in undifferentiated/unpolarized vs. differentiated/polarized cells. While basal localization of the S1423N mutant gets corrected in the differentiated glia, its Cu(I) responsive anterograde and retrograde trafficking behavior is not identical to the wild-type. But localization and trafficking properties are completely rescued for the S1426I and T1434M mutants in the differentiated cells. Comprehensive meta-analysis on the effect of the reported C-terminal mutations on patient phenotype and cultured cells demonstrate discrete regions having distinct effects. While mutations in the proximal C-terminus affect ATP7B stability, the present study shows that the distal region dictates cell-specific Trans Golgi Network (TGN) localization and exit. The localization and export properties are corrected in the differentiated cells, which is a plausible mechanism for the milder phenotype exhibited by these mutations. It highlights the critical role of the C-terminus in cell-specific TGN retention and exit of ATP7B.

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.