Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited.

S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity. We further carry out structural comparisons employing the recently determined crystal structure of oxetanocin-A biosynthetic enzyme OxsB, the first three-dimensional structural data on a Cbl-dependent AdoMet radical enzyme. We find that the structural motifs responsible for housing the AdoMet radical machinery are largely conserved, whereas the motifs responsible for binding additional cofactors are much more varied.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Copper regulates the host innate immune response against bacterial infection via activation of ALPK1 kinase.

Copper is an essential trace element for the human body, and its requirement for optimistic immune functions has been recognized for decades. How copper is involved in the innate immune pathway, however, remains to be clarified. Here, we report that copper serves as a signal molecule to regulate the kinase activity of alpha-kinase 1 (ALPK1), a cytosolic pattern-recognition receptor (PRR), and therefore promotes host cell defense against bacterial infection. We show that in response to infection, host cells actively accumulate copper in the cytosol, and the accumulated cytosolic copper enhances host cell defense against evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, we demonstrate that copper activates the innate immune pathway of host cells in an ALPK1-dependent manner. Further mechanistic studies reveal that copper binds to ALPK1 directly and is essential for the kinase activity of this cytosolic PRR. Moreover, the binding of copper to ALPK1 enhances the sensitivity of ALPK1 to the bacterial metabolite ADP-heptose and eventually prompts host cells to elicit an enhanced immune response during bacterial infection. Finally, using a zebrafish in vivo model, we show that a copper-treated host shows an increased production of proinflammatory cytokines, enhanced recruitment of phagosome cells, and promoted bacterial clearance. Our findings uncover a previously unrecognized role of copper in the modulation of host innate immune response against bacterial pathogens and advance our knowledge on the cross talk between cytosolic copper homeostasis and immune system.

Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome.

The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs.

Heme metabolism in nonerythroid cells.

Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins." Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In nonerythroid cells, where cytosolic heme concentrations are 2 to 3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here, we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology.

GPDOCK: highly accurate docking strategy for metalloproteins based on geometric probability.

Accurately predicting the interaction modes for metalloproteins remains extremely challenging in structure-based drug design and mechanism analysis of enzymatic catalysis due to the complexity of metal coordination in metalloproteins. Here, we report a docking method for metalloproteins based on geometric probability (GPDOCK) with unprecedented accuracy. The docking tests of 10 common metal ions with 9360 metalloprotein-ligand complexes demonstrate that GPDOCK has an accuracy of 94.3% in predicting binding pose. What is more, it can accurately realize the docking of metalloproteins with ligand when one or two water molecules are engaged in the metal ion coordination. Since GPDOCK only depends on the three-dimensional structure of metalloprotein and ligand, structure-based machine learning model is employed for the scoring of binding poses, which significantly improves computational efficiency. The proposed docking strategy can be an effective and efficient tool for drug design and further study of binding mechanism of metalloproteins. The manual of GPDOCK and the code for the logistical regression model used to re-rank the docking results are available at https://github.com/wangkai-zhku/GPDOCK.git.

Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion.

The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the NiI species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

Bacterial hemophilin homologs and their specific type eleven secretor proteins have conserved roles in heme capture and are diversifying as a family.

Cellular life relies on enzymes that require metals, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogens Neisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii, and Haemophilus influenzae. Experimentally verified T11SS-dependent cargo include transferrin-binding protein B (TbpB), the hemophilin homologs heme receptor protein C (HrpC), hemophilin A (HphA), the immune evasion protein factor-H binding protein (fHbp), and the host symbiosis factor nematode intestinal localization protein C (NilC). Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explored the ligand binding ability of those hemophilin cargo homologs. In vivo expression in Escherichia coli of hemophilin homologs revealed that each is secreted in a specific manner by its cognate T11SS protein. Sequence analysis and structural modeling suggest that all hemophilin homologs share an N-terminal ligand-binding domain with the same topology as the ligand-binding domains of the Haemophilus haemolyticus heme binding protein (Hpl) and HphA. We term this signature feature of this group of proteins the hemophilin ligand-binding domain. Network analysis of hemophilin homologs revealed five subclusters and representatives from four of these showed variable heme-binding activities, which, combined with sequence-structure variation, suggests that hemophilins are diversifying in function.IMPORTANCEThe secreted protein hemophilin and its homologs contribute to the survival of several bacterial symbionts within their respective host environments. Here, we compared taxonomically diverse hemophilin homologs and their paired Type 11 secretion systems (T11SS) to determine if heme binding and T11SS secretion are conserved characteristics of this family. We establish the existence of divergent hemophilin sub-families and describe structural features that contribute to distinct ligand-binding behaviors. Furthermore, we demonstrate that T11SS are specific for their cognate hemophilin family cargo proteins. Our work establishes that hemophilin homolog-T11SS pairs are diverging from each other, potentially evolving into novel ligand acquisition systems that provide competitive benefits in host niches.

The mitochondrial amidoxime reducing component-from prodrug-activation mechanism to drug-metabolizing enzyme and onward to drug target.

The mitochondrial amidoxime-reducing component (mARC) is one of five known molybdenum enzymes in eukaryotes. mARC belongs to the MOSC domain superfamily, a large group of so far poorly studied molybdoenzymes. mARC was initially discovered as the enzyme activating N-hydroxylated prodrugs of basic amidines but has since been shown to also reduce a variety of other N-oxygenated compounds, for example, toxic nucleobase analogs. Under certain circumstances, mARC might also be involved in reductive nitric oxide synthesis through reduction of nitrite. Recently, mARC enzymes have received a lot of attention due to their apparent involvement in lipid metabolism and, in particular, because many genome-wide association studies have shown a common variant of human mARC1 to have a protective effect against liver disease. The mechanism linking mARC enzymes with lipid metabolism remains unknown. Here, we give a comprehensive overview of what is currently known about mARC enzymes, their substrates, structure, and apparent involvement in human disease.

Hemes on a string: insights on the functional mechanisms of PgcA from Geobacter sulfurreducens.

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.

Rubredoxin 1 promotes the proper folding of D1 and is not required for heme b559 assembly in Chlamydomonas photosystem II.

Photosystem II (PSII), the water:plastoquinone oxidoreductase of oxygenic photosynthesis, contains a heme b559 iron whose axial ligands are provided by histidine residues from the α (PsbE) and ß (PsbF) subunits. PSII assembly depends on accessory proteins that facilitate the step-wise association of its protein and pigment components into a functional complex, a process that is challenging to study due to the low accumulation of assembly intermediates. Here, we examined the putative role of the iron[1Fe-0S]-containing protein rubredoxin 1 (RBD1) as an assembly factor for cytochrome b559, using the RBD1-lacking 2pac mutant from Chlamydomonas reinhardtii, in which the accumulation of PSII was rescued by the inactivation of the thylakoid membrane FtsH protease. To this end, we constructed the double mutant 2pac ftsh1-1, which harbored PSII dimers that sustained its photoautotrophic growth. We purified PSII from the 2pac ftsh1-1 background and found that α and ß cytochrome b559 subunits are still present and coordinate heme b559 as in the WT. Interestingly, immunoblot analysis of dark- and low light-grown 2pac ftsh1-1 showed the accumulation of a 23-kDa fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage of PSII. Its cleavage occurs in the vicinity of a nonheme iron which binds to PSII on its electron acceptor side. Altogether, our findings demonstrate that RBD1 is not required for heme b559 assembly and point to a role for RBD1 in promoting the proper folding of D1, possibly via delivery or reduction of the nonheme iron during PSII assembly.

Apo-metallothionein-3 cooperatively forms tightly compact structures under physiological conditions.

Metallothioneins (MTs) are essential mammalian metal chaperones. MT isoform 1 (MT1) is expressed in the kidneys and isoform 3 (MT3) is expressed in nervous tissue. For MTs, the solution-based NMR structure was determined for metal-bound MT1 and MT2, and only one X-ray diffraction structure on a crystallized mixed metal-bound MT2 has been reported. The structure of solution-based metalated MT3 is partially known using NMR methods; however, little is known about the fluxional de novo apo-MT3 because the structure cannot be determined by traditional methods. Here, we used cysteine modification coupled with electrospray ionization mass spectrometry, denaturing reactions with guanidinium chloride, stopped-flow methods measuring cysteine modification and metalation, and ion mobility mass spectrometry to reveal that apo-MT3 adopts a compact structure under physiological conditions and an extended structure under denaturing conditions, with no intermediates. Compared with apo-MT1, we found that this compact apo-MT3 binds to a cysteine modifier more cooperatively at equilibrium and 0.5 times the rate, providing quantitative evidence that many of the 20 cysteines of apo-MT3 are less accessible than those of apo-MT1. In addition, this compact apo-MT3 can be identified as a distinct population using ion mobility mass spectrometry. Furthermore, proposed structural models can be calculated using molecular dynamics methods. Collectively, these findings provide support for MT3 acting as a noninducible regulator of the nervous system compared with MT1 as an inducible scavenger of trace metals and toxic metals in the kidneys.

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor.

In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H. pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states. Nickel binding slows down conformational exchange between these states and shifts the equilibrium toward the binding-competent states. The protein then becomes stabilized in a cis conformation upon binding the ureA promoter. Here, we investigate how nickel binding creates this response and how it is transmitted to the DNA-binding domains. Through mutagenesis, DNA-binding studies, and computational methods, the allosteric response to nickel was found to be propagated from the nickel-binding sites to the DNA-binding domains via the ß-sheets of the metal-binding domain and a network of residues at the inter-domain interface. Our computational results suggest that nickel binding increases protein rigidity to slow down the conformational exchange. A thymine base in the ureA promoter sequence, known to be critical for high affinity DNA binding by HpNikR, was also found to be important for the allosteric response, while a modified version of this promoter further highlighted the importance of the DNA sequence in modulating the response. Collectively, our results provide insights into regulation of a key protein for H. pylori survival.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator.

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of dinitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at noncryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the fumarate and nitrate reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR-binding sites. By coupling size-exclusion chromatography-anSAXS with full-spectrum UV-Vis analysis, we show that the [4Fe-4S] cluster-containing dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

GASS-Metal: identifying metal-binding sites on protein structures using genetic algorithms.

Metals are present in >30% of proteins found in nature and assist them to perform important biological functions, including storage, transport, signal transduction and enzymatic activity. Traditional and experimental techniques for metal-binding site prediction are usually costly and time-consuming, making computational tools that can assist in these predictions of significant importance. Here we present Genetic Active Site Search (GASS)-Metal, a new method for protein metal-binding site prediction. The method relies on a parallel genetic algorithm to find candidate metal-binding sites that are structurally similar to curated templates from M-CSA and MetalPDB. GASS-Metal was thoroughly validated using homologous proteins and conservative mutations of residues, showing a robust performance. The ability of GASS-Metal to identify metal-binding sites was also compared with state-of-the-art methods, outperforming similar methods and achieving an MCC of up to 0.57 and detecting up to 96.1% of the sites correctly. GASS-Metal is freely available at https://gassmetal.unifei.edu.br. The GASS-Metal source code is available at https://github.com/sandroizidoro/gassmetal-local.

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Deciphering the dual nature of nesfatin-1: a tale of zinc ion's Janus-faced influence.

BACKGROUND: Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2). RESULTS: Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule. CONCLUSIONS: In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2.

Peptides and metal ions: A successful marriage for developing artificial metalloproteins.

The mutual relationship between peptides and metal ions enables metalloproteins to have crucial roles in biological systems, including structural, sensing, electron transport, and catalytic functions. The effort to reproduce or/and enhance these roles, or even to create unprecedented functions, is the focus of protein design, the first step toward the comprehension of the complex machinery of nature. Nowadays, protein design allows the building of sophisticated scaffolds, with novel functions and exceptional stability. Recent progress in metalloprotein design has led to the building of peptides/proteins capable of orchestrating the desired functions of different metal cofactors. The structural diversity of peptides allows proper selection of first- and second-shell ligands, as well as long-range electrostatic and hydrophobic interactions, which represent precious tools for tuning metal properties. The scope of this review is to discuss the construction of metal sites in de novo designed and miniaturized scaffolds. Selected examples of mono-, di-, and multi-nuclear binding sites, from the last 20 years will be described in an effort to highlight key artificial models of catalytic or electron-transfer metalloproteins. The authors' goal is to make readers feel like guests at the marriage between peptides and metal ions while offering sources of inspiration for future architects of innovative, artificial metalloproteins.

Decarbromodiphenyl ether exposure promotes migration of triple-negative breast cancer cells through miR-221 in extracellular vesicles. / åæº´è”è‹¯é†šæš´éœ²é€šè¿‡èƒžå¤–å›Šæ³¡è£ è½½miR-221ä¿ƒè¿›ä¸‰é˜´æ€§ä¹³è ºç™Œç»†èƒžè½¬ç§».

OBJECTIVES: To investigate the effect of decarbromodiphenyl ether (BDE-209) exposure on the migration ability of triple-negative breast cancer (TNBC) cells and to explore the underlying mechanism. METHODS: Human TNBC MDA-MB-231 cells were divided into blank control group and BDE-209 exposure groups (treated with 0.02, 0.20, 2.00, 20.00 and 200.00 ng/mL BDE-209 in high glucose DMEM). Extracellular vehicles (EVs) secreted by MDA-MB-231 cells were isolated by differential ultracentrifugation. Transmission electron microscopy (SEM), nanoparticle tracking analysis (NTA) and Western blotting were performed to characterize the EVs. The effect of the EVs induced by BDE-209 exposure (EVs-BDE-209) on the migration and invasion of MDA-MB-231 cells was detected by wound-healing assay and Transwell test. qRT-PCR was used to measure the miR-221 level in EVs-BDE-209. The expression of MMP9 in MDA-MB-231 cells was determined by Western blotting. RESULTS: Compared with the blank control, BDE-209 exposure increased the tumor cell-derived EVs in dose-dependent manner. The MDA-MB-231 cells co-cultured with EVs released by 200.00 ng/mL BDE-209 exposure showed an 86% increase in cell migration rate, a 1.32-fold higher number of membrane-penetrating cells, a 2.71-fold higher expression level of miR-221, and a 1.62-fold higher expression level of MMP9 compared with the blank control group (all P<0.05). While transfection with anti-miR-221 antibody to decrease miR-221 level in EVs significantly reversed the increased invasion ability of the MDA-MB-231 cells treated with EVs-BDE-209. CONCLUSIONS: BDE-209 exposure may promote metastasis potential of MDA-MB-231 cells via EVs-BDE-209 transmitted miR-221.