Metallomics最新文献

This work aims to evaluate the size and lability of Cu and Zn bound to proteins in the cytosol of fish liver of Oreochromis niloticus by employing solid-phase extraction (SPE), diffusive gradients in thin films (DGT), and ultrafiltration (UF). SPE was carried out using Chelex-100. DGT containing Chelex-100 as binding agent was employed. Analyte concentrations were determined by ICP-MS. Total Cu and Zn concentrations in cytosol (1 g of fish liver in 5 ml of Tris-HCl) ranged from 39.6 to 44.3 ng ml-1 and 1498 to 2106 ng ml-1, respectively. Data from UF (10-30 kDa) suggested that Cu and Zn in cytosol were associated with ∼70% and 95%, respectively, with high-molecular-weight proteins. Cu-metallothionein was not selectively detected (although 28% of Cu was associated with low-molecular-weight proteins). However, information about the specific proteins in the cytosol will require coupling UF with organic mass spectrometry. Data from SPE showed the presence of labile Cu species of ∼17%, while the fraction of labile Zn species was >55%. However, data from DGT suggested a fraction of labile Cu species only of 7% and a labile Zn fraction of 5%. This data, as compared with previous data from literature, suggests that the DGT technique gave a more plausible estimation of the labile pool of Zn and Cu in cytosol. The combination of results from UF and DGT is capable of contributing to the knowledge about the labile and low-molecular pool of Cu and Zn.

Loss-of-function mutations in SLC30A10 induce hereditary manganese (Mn)-induced neuromotor disease in humans. We previously identified SLC30A10 to be a critical Mn efflux transporter that controls physiological brain Mn levels by mediating hepatic and intestinal Mn excretion in adolescence/adulthood. Our studies also revealed that in adulthood, SLC30A10 in the brain regulates brain Mn levels when Mn excretion capacity is overwhelmed (e.g. after Mn exposure). But, the functional role of brain SLC30A10 under physiological conditions is unknown. We hypothesized that, under physiological conditions, brain SLC30A10 may modulate brain Mn levels and Mn neurotoxicity in early postnatal life because body Mn excretion capacity is reduced in this developmental stage. We discovered that Mn levels of pan-neuronal/glial Slc30a10 knockout mice were elevated in specific brain regions (thalamus) during specific stages of early postnatal development (postnatal day 21), but not in adulthood. Furthermore, adolescent or adult pan-neuronal/glial Slc30a10 knockouts exhibited neuromotor deficits. The neuromotor dysfunction of adult pan-neuronal/glial Slc30a10 knockouts was associated with a profound reduction in evoked striatal dopamine release without dopaminergic neurodegeneration or changes in striatal tissue dopamine levels. Put together, our results identify a critical physiological function of brain SLC30A10-SLC30A10 in the brain regulates Mn levels in specific brain regions and periods of early postnatal life, which protects against lasting deficits in neuromotor function and dopaminergic neurotransmission. These findings further suggest that a deficit in dopamine release may be a likely cause of early-life Mn-induced motor disease.

Protein reactions play important roles in the mechanism of action of cisplatin. In this work, we found that cisplatin is highly reactive to the RING finger domain of RNF11, a key protein involved in tumorigenesis and metastasis. The results show that cisplatin binds to RNF11 at the zinc coordination site and leads to zinc ejection from the protein. The formation of S-Pt(II) coordination and Zn(II) ions release have been confirmed by UV-vis spectrometry using zinc dye and thiol agent, showing reducing the contents of thiol groups while forming S-Pt bonds and releasing zinc ions. Electrospray ionization-mass spectrometry measurement indicates that each RNF11 can bind up to three platinum atoms. Kinetical analysis shows a reasonable platination rate of RNF11 with t1/2 ∼ 3 h. CD, nuclear magnetic resonance, and gel electrophoresis measurements indicate that the cisplatin reaction causes protein unfolding and oligomerization of RNF11. Pull-down assay confirms that the platination of RNF11 interferes with the protein interaction of RNF11 with UBE2N, a key step of the functionalization of RNF11. Furthermore, Cu(I) was found to promote the platination of RNF11, which could lead to increased protein reactivity to cisplatin in tumor cells with high copper levels. These results indicate that the platination-induced zinc release of RNF11 disrupts the protein structure and interferes with its functions.

The diffuse and renewed use of silver as antimicrobial agent has caused the development of resistance to silver ions in some bacterial strains, posing a serious threat for health systems. In order to cast light on the mechanistic features of resistance, here, we aimed to understand how silver interacts with the periplasmic metal-binding protein SilE which is engaged in bacterial silver detoxification. This aim was addressed by studying two peptide portions of SilE sequence (SP2 and SP3) that contain the putative motifs involved in Ag+ binding. We demonstrate that SP2 model peptide is involved in silver binding through its histidine and methionine residues in the two HXXM binding sites. In particular, the first binding site is supposed to bind the Ag+ ion in a linear fashion, while the second binding site complexes the silver ion in a distorted trigonal planar fashion. We propose a model where the SP2 peptide binds two silver ions when the concentration ratio Ag+/SP2 is ≥10.0. We also suggest that the two binding sites of SP2 have different affinity for silver. This evidence comes from the change in the path direction of the Nuclear Magnetic Resonance (NMR) cross-peaks upon the addition of Ag+. Here, we report the conformational changes of SilE model peptides occurring upon silver binding, monitored at a deep level of molecular details. This was addressed by a multifaceted approach, combining NMR, circular dichroism, and mass spectrometry experiments.

Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry (MS/MS) of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry (MS)-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme-binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin, and finally elution and identification by MS. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in an SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization, and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role of heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

Understanding of how mercury species cause cellular impairments at the molecular level is critical for explaining the detrimental effects of mercury exposure on the human body. Previous studies have reported that inorganic and organic mercury compounds can induce apoptosis and necrosis in a variety of cell types, but more recent advances reveal that mercuric mercury (Hg2+) and methylmercury (CH3Hg+) may result in ferroptosis, a distinct form of programmed cell death. However, it is still unclear which protein targets are responsible for ferroptosis induced by Hg2+ and CH3Hg+. In this study, human embryonic kidney 293T cells were used to investigate how Hg2+ and CH3Hg+ trigger ferroptosis, given their nephrotoxicity. Our results demonstrate that glutathione peroxidase 4 (GPx4) plays a key role in lipid peroxidation and ferroptosis in renal cells induced by Hg2+ and CH3Hg+. The expression of GPx4, the only lipid repair enzyme in mammal cells, was downregulated in response to Hg2+ and CH3Hg+ stress. More importantly, the activity of GPx4 could be markedly inhibited by CH3Hg+, owing to the direct binding of the selenol group (-SeH) in GPx4 to CH3Hg+. Selenite supplementation was demonstrated to enhance the expression and activity of GPx4 in renal cells, and consequently relieve the cytotoxicity of CH3Hg+, suggesting that GPx4 is a crucial modulator implicated in the Hg-Se antagonism. These findings highlight the importance of GPx4 in mercury-induced ferroptosis, and provide an alternative explanation for how Hg2+ and CH3Hg+ induce cell death.

The increased use of antimicrobial compounds such as copper into nanoparticles changes how living cells interact with these novel materials. The increased use of antimicrobial nanomaterials combats infectious disease and food spoilage. Fungal infections are particularly difficult to treat because of the few druggable targets, and Saccharomyces cerevisiae provides an insightful model organism to test these new materials. However, because of the novel characteristics of these materials, it is unclear how these materials interact with living cells and if resistance to copper-based nanomaterials could occur. Copper nanoparticles built on carboxymethylcellulose microfibril strands with copper (CMC-Cu) are a promising nanomaterial when imported into yeast cells and induce cell death. The α-arrestins are cargo adaptors that select which molecules are imported into eukaryotic cells. We screened α-arrestins mutants and identified Aly2, Rim8, and Rog3 α-arrestins, which are necessary for the internalization of CMC-Cu nanoparticles. Internal reactive oxygen species in these mutants were lower and corresponded to the increased viability in the presence of CMC-Cu. Using lattice light-sheet microscopy on live cells, we determined that CMC-Cu were imported into yeast within 30 min of exposure. Initially, the cytoplasmic pH decreased but returned to basal level 90 min later. However, there was heterogeneity in response to CMC-Cu exposure, which could be due to the heterogeneity of the particles or differences in the metabolic states within the population. When yeast were exposed to sublethal concentrations of CMC-Cu no resistance occurred. Internalization of CMC-Cu increases the potency of these antimicrobial nanomaterials and is likely key to preventing fungi from evolving resistance.

The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of β-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.