Using a chemical genetic screen to enhance our understanding of the antimicrobial properties of copper

The competitive toxic and stress inducing nature of copper necessitates systems that sequester and export this metal from the cytoplasm of bacterial cells. Several predicted mechanisms of toxicity include the production of reactive oxygen species, thiol depletion, DNA and iron-sulfur cluster disruption. Accompanying these mechanisms include pathways of homeostasis such as chelation, oxidation, and transport. Still, the mechanisms of copper resistance and sensitivity are not fully understood. Furthermore, studies fail to recognize that the response to copper is likely a result of numerous mechanisms, as in the case for homeostasis, in which proteins and enzymes work as a collective to maintain appropriate copper concentrations. In this study we used the Keio collection, an array of 3985 Escherichia coli mutants, each with a deleted non-essential gene, to gain a better understanding of prolonged copper exposure. In short, we recovered two copper homeostatic gene and genes involved in transporting and assembling to be involved in mediating prolonged copper stress under the conditions assessed. The gene coding for the protein TolC was uncovered as a sensitive hit and we demonstrated that tolC, an outer membrane efflux channel, is key in mitigating copper sensitivity. Additionally, the activity of tRNA processing was enriched and the deletion of several proteins involved in import generated copper tolerance. Lastly, key genes belonging to central carbon metabolism and nicotinamide adenine dinucleotide biosynthesis were uncovered as tolerant hits. Overall, this study shows that copper sensitivity and tolerance are a result of numerous mechanisms acting in combination within the cell.

Lipidomics reveals perturbations in the liver lipid profile of iron overloaded mice

Iron overload is an important contributor to disease. The liver, the major site of iron storage in the body, is a key organ impacted by iron overload. While several studies have reported perturbations in liver lipids in iron overload, it is not clear, on a global scale, how individual liver lipid ions are altered. Here, we used lipidomics to study the changes in hepatic lipid ions in iron-overloaded mice. Iron overload was induced by daily intraperitoneal injections of 100mg/kg body weight iron dextran for one week. Iron overload was verified by serum markers of iron status, liver iron quantitation, and Perls’ stain. Compared with the control group, the serum of iron-overload mice exhibited low levels of urea nitrogen and high-density lipoprotein (HDL), and high concentrations of total bile acid, low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), suggestive of liver injury. Moreover, iron overload disrupted liver morphology, induced reactive oxygen species (ROS) production, reduced superoxide dismutase (SOD) activity, caused lipid peroxidation, and led to DNA fragmentation. Iron overload altered the overall composition of lipid ions in the liver, with significant changes in over 100 unique lipid ions. Notably, iron overload selectively increased the overall abundance of glycerolipids and changed the composition of glycerophospholipids and sphingolipids. This study, one of the first to report iron-overload induced lipid alterations on a global lipidomics scale, provides early insight into lipid ions that may be involved in iron overload-induced pathology.

Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Microbes: A potential tool for selenium biofortification

Selenium (Se) is a component of many enzymes and indispensable for human health due to its characteristics of reducing oxidative stress and enhancing immunity. Human beings take Se mainly from Se-containing crops. Taking measures to biofortify crops with Se may lead to improved public health. Se accumulation in plants mainly depends on the content and bioavailability of Se in soil. Beneficial microbes may change the chemical form and bioavailability of Se. This review highlights the potential role of microbes in promoting Se uptake and accumulation in crops and the related mechanisms. The potential approaches of microbial enhancement of Se biofortification can be summarized in the following four aspects: (1) microbes alter soil properties and impact the redox chemistry of Se to improve the bioavailability of Se in soil; (2) beneficial microbes regulate root morphology and stimulate the development of plants through the release of certain secretions, facilitating Se uptake in plants; (3) microbes upregulate the expression of certain genes and proteins that are related to Se metabolism in plants; (4) the inoculation of microbes give rise to the generation of certain metabolites in plants contributing to Se absorption. Considering the ecological safety and economic feasibility, microbial enhancement is a potential tool for Se biofortification. For further study, the recombination and establishment of synthesis microbes is of potential benefit in Se-enrichment agriculture.

Zinc Trafficking 1. Probing the Roles of Proteome, Metallothionein, and Glutathione

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that non-specific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione. In agreement, inclusion of glutathione accelerated the reaction in a concentration-dependent manner. The implications of abundant high affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with glutathione in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

Mechanisms of irreversible aquaporin-10 inhibition by organogold compounds studied by combined biophysical methods and atomistic simulations

The inhibition of glycerol permeation via human aquaporin-10 (hAQP10) by organometallic gold complexes has been studied by fluorescence stopped-flow spectroscopy, and its mechanism has been described using molecular modelling and atomistic simulations. The most effective hAQP10 inhibitors are cyclometalated Au(III) C^N compounds known to efficiently react with cysteine residues leading to the formation of irreversible C—S bonds. Functional assays also demonstrate the irreversibility of the binding to hAQP10 by the organometallic complexes. The obtained computational results by metadynamics show that the local arylation of Cys209 in hAQP10 by one of the gold inhibitors is mapped into a global change of the overall free energy of glycerol translocation across the channel. Our study further pinpoints the need to understand the mechanism of glycerol and small molecule permeation as a combination of local structural motifs and global pore conformational changes, which are taking place on the scale of the translocation process and whose study therefore, require sophisticated molecular dynamics strategies.

Trace elements in dried blood spots as potential discriminating features for metabolic disorders diagnosis in new-borns

Trace elements in dried blood spots (DBS) from new-born were determined by laser ablation (LA) coupled with inductively coupled mass spectrometry (ICP-MS), and data was subjected to chemometric evaluation in an attempt to classify healthy new-born and new-born suffering metabolic disorders. Unsupervised [principal component analysis (PCA), and cluster analysis (CA)], and supervised [linear discriminant analysis (LDA), and soft independent modelling by class analogy (SIMCA)] pattern recognition techniques were used as classification techniques. PCA and CA have shown a clear tendency to form two groups (healthy and new-born suffering metabolic disorders). LDA and SIMCA have predicted that 90.5 and 83.9% of original grouped healthy new-born cases were correctly classified by LDA and SIMCA, respectively. In addition, these percentages were 97.6% (LDA) and 80.6% (SIMCA) for DBSs from new-born suffering metabolic disorders. However, SIMCA has only detected one misclassified DBS from the healthy group, and the lower percentage is attributed to four DBDs from healthy new-borns group and five DBSs from new-borns with disorders which were found to be as belonging to both categories (healthy new-borns and new-borns with disorders) in the training set. LDA also gave a % of grouped maple syrup urine disease (MSUD) cases correctly classified of 100%, although the percentage fells to 66.7% when classifying phenylketonuria (PKU) cases. Finally, essential elements such as Fe, K, Rb, and Zn were found to be matched (correlated) with the concentration of amino acids such as phenylalanine, valine and leucine, biomarkers linked with MSUD and PKU diseases.

The physiology and evolution of microbial selenium metabolism

Selenium is an essential trace element whose compounds are widely metabolized by organisms from all three domains of life. Moreover, phylogenetic evidence indicates that selenium species, along with iron, molybdenum, tungsten, and nickel, were metabolized by the last universal common ancestor (LUCA) of all cellular lineages, primarily for the synthesis of the 21st amino acid selenocysteine. Thus, selenium metabolism is both environmentally ubiquitous and a physiological adaptation of primordial life. Selenium metabolic reactions comprise reductive transformations both for assimilation into macromolecules and dissimilatory reduction of selenium oxyanions and elemental selenium during anaerobic respiration. This review offers a comprehensive overview of the physiology and evolution of both assimilatory and dissimilatory selenium metabolism in bacteria and archaea, highlighting mechanisms of selenium respiration. This includes a thorough discussion of our current knowledge of the physiology of selenocysteine synthesis and incorporation into proteins in bacteria obtained from structural biology. Additionally, this is the first comprehensive discussion in a review of the incorporation of selenium into the tRNA nucleoside 5-methylaminomethyl-2-selenouridine and as an inorganic cofactor in certain molybdenum hydroxylase enzymes. Throughout, conserved mechanisms and derived features of selenium metabolism in both domains are emphasized and discussed within the context of the global selenium biogeochemical cycle.

Structurally restricted Bi(III) metallation of apo-βMT1a: metal-induced tangling

Non-toxic bismuth salts are used in anti-ulcer medications and to protect against nephrotoxicity from anti-cancer drugs. Bismuth salts also induce metallothionein (MT), a metal-binding protein that lacks a formal secondary structure. We report the impact on the metallation properties of Bi(III) to the 9-cysteine β fragment of MT as a function of cysteine accessibility using electrospray ionization mass spectrometry. At pH 7.4, Bi2βMT formed cooperatively. Cysteine modification shows that each Bi(III) was terminally bound to 3 cysteinyl thiolates. Non-cooperative Bi(III) binding was observed at pH 2.3, where cysteine accessibility is increased. However, competition from H4EDTA inhibited Bi(III) binding. When GdmCl, a well-known denaturing agent, was used to increase cysteine accessibility of the apoβMT at pH 7.4, a greater fraction of Bi3βMT formed using all 9 cysteines. The change in binding profile and equilibrium of Bi2βMT was determined as a function of acidification, which changed as a result of competition with H4EDTA. There was no Bi(III) transfer between Bi2βMT, Cd3βMT, and Zn3βMT. This lack of metal exchange and the resistance towards binding the third Bi(III) suggests a rigidity in the Bi2βMT binding sites that inhibits Bi(III) mobility. These experiments emphasize the conformational control of metallation that results in substantially different metallated products: at pH 7.4 (many cysteines buried) Bi2βMT, whereas at pH 7.4 (all cysteines accessible) enhanced formation of Bi3βMT. These data suggest that the addition of the first 2 Bi(III) cross-link the protein, blocking access to the remaining 3 cysteines for the third Bi(III), as a result of tangle formation.

Chemistry and Biology of Ferritin

Iron is an essential element required by cells and has been described as a key player in ferroptosis. Ferritin operates as a fundamental iron storage protein in cells forming multimeric assemblies with crystalline iron cores. We discuss the latest findings on ferritin structure and activity and its link to cell metabolism and ferroptosis. The chemistry of iron, including its oxidations states, is important for its biological functions, its reactivity and the biology of ferritin. Ferritin can be localized in different cellular compartments and secreted by cells with a variety of functions depending on its spatial context. Here, we discuss how cellular ferritin localization is tightly linked to its function in a tissue-specific manner, and how impairment of iron homeostasis is implicated in diseases including cancer and COVID-19. Ferritin is a potential biomarker and we discuss latest research where it has been employed for imaging purposes and drug delivery.