Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Altering relative metal-binding affinities in multifunctional Metallochaperones for mutant p53 reactivation.

The p53 protein plays a major role in cancer prevention, and over 50% of cancer diagnoses can be attributed to p53 malfunction. p53 incorporates a structural Zn site that is required for proper protein folding and function, and in many cases point mutations can result in loss of the Zn2+ ion, destabilization of the tertiary structure, and eventual amyloid aggregation. Herein, we report a series of compounds designed to act as small molecule stabilizers of mutant p53, and feature Zn-binding fragments to chaperone Zn2+ to the metal depleted site and restore wild-type (WT) function. Many Zn metallochaperones (ZMCs) have been shown to generate intracellular reactive oxygen species (ROS), likely by chelating redox-active metals such as Fe2+/3+ and Cu+/2+ and undergoing associated Fenton chemistry. High levels of ROS can result in off-target effects and general toxicity, and thus, careful tuning of ligand Zn2+ affinity, in comparison to the affinity for other endogenous metals, is important for selective mutant p53 targeting. In this work we show that by using carboxylate donors in place of pyridine we can change the relative Zn2+/Cu2+ binding ability in a series of ligands, and we investigate the impact of donor group changes on metallochaperone activity and overall cytotoxicity in two mutant p53 cancer cell lines (NUGC3 and SKGT2).

In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation.

The importance of cellular low molecular weight ligands in metalloenzyme maturation is largely unexplored. Maturation of NiSOD requires post-translational N-terminal processing of the proenzyme, SodN, by its cognate protease, SodX. Here we provide evidence for the participation of L-histidine in the protease-dependent maturation of nickel-dependent superoxide dismutase (NiSOD) from Streptomyces coelicolor. In vitro studies using purified proteins cloned from S. coelicolor and overexpressed in E. coli support a model where a ternary complex formed between the substrate (SodN), the protease (SodX) and L-Histidine creates a novel Ni-binding site that is capable of the N-terminal processing of SodN and specifically incorporates Ni into the apo-NiSOD product. Thus, L-Histidine serves many of the functions associated with a metallochaperone or, conversely, eliminates the need for a metallochaperone in NiSOD maturation.

The SlyD metallochaperone targets iron-sulfur biogenesis pathways and the TCA cycle.

IMPORTANCE: Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis/trans-isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori, measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies.

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase (copA) in survival in a mouse lung infection model. Here, we demonstrate that H. influenzae copA (HI0290) is responsible for copper homeostasis involving the merR-type regulator, cueR, as well as six tandem copies of the metallochaperone gene, copZ. Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. Nontypeable H. influenzae (NTHi) clinical isolate NT127 has the same locus organization but with three copies of copZ. We showed that expression of the NTHi copZA operon is activated by copper under the regulatory control of CueR. NTHi single copA and copZ mutants and, especially, the double deletion copZA mutant exhibited decreased copper tolerance, and the ΔcopZA mutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase (copA) alone and deleted of both the ATPase and chaperones (copZ1-3) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation of cop locus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that the cop system encodes an important countermeasure to alleviate copper toxicity.

Copper chaperone antioxidant 1: multiple roles and a potential therapeutic target.

Copper (Cu) was recently demonstrated to play a critical role in cellular physiological and biochemical processes, including energy production and maintenance, antioxidation and enzymatic activity, and signal transduction. Antioxidant 1 (ATOX1), a chaperone of Cu previously named human ATX1 homologue (HAH1), has been found to play an indispensable role in maintaining cellular Cu homeostasis, antioxidative stress, and transcriptional regulation. In the past decade, it has also been found to be involved in a variety of diseases, including numerous neurodegenerative diseases, cancers, and metabolic diseases. Recently, increasing evidence has revealed that ATOX1 is involved in the regulation of cell migration, proliferation, autophagy, DNA damage repair (DDR), and death, as well as in organism development and reproduction. This review summarizes recent advances in the research on the diverse physiological and cytological functions of ATOX1 and the underlying mechanisms of its action in human health and diseases. The potential of ATOX1 as a therapeutic target is also discussed. This review aims to pose unanswered questions related to ATOX1 biology and explore the potential use of ATOX1 as a therapeutic target.

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C.

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery.

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(ß,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

Metallochaperone OsHIPP9 is involved in the retention of cadmium and copper in rice.

Metallochaperones are a unique class of proteins that play crucial roles in metal homoeostasis and detoxification. However, few metallochaperones have been functionally characterised in rice. Heterologous expression of Heavy metal-associated Isoprenylated Plant Protein 9 (OsHIPP9), a metallochaperone, altered yeast tolerance to cadmium (Cd) and copper (Cu). We investigated the physiological role of OsHIPP9 in rice. OsHIPP9 was primarily expressed in the root exodermis and xylem region of enlarged vascular bundles (EVB) at nodes. KO of OsHIPP9 increased the Cd concentrations of the upper nodes and panicle, but decreased Cd in expanded leaves. KO of OsHIPP9 decreased Cu uptake and accumulation in rice. Constitutive OX of OsHIPP9 increased Cd and Cu accumulation in aboveground tissues and brown rice. OsHIPP9 showed binding capacity for Cd and Cu. We propose that OsHIPP9 has dual metallochaperone roles, chelating Cd in the xylem region of EVB for Cd retention in the nodes and chelating Cu in rice roots to aid Cu uptake.

Copper coordination states affect the flexibility of copper Metallochaperone Atox1: Insights from molecular dynamics simulations.

Copper is an essential element in nature but in excess, it is toxic to the living cell. The human metallochaperone Atox1 participates in copper homeostasis and is responsible for copper transmission. In a previous multiscale simulation study, we noticed a change in the coordination state of the Cu(I) ion, from 4 bound cysteine residues to 3, in agreement with earlier studies. Here, we perform and analyze classical molecular dynamic simulations of various coordination states: 2, 3, and 4. The main observation is an increase in protein flexibility as a result of a decrease in the coordination state. In addition, we identified several populated conformations that correlate well with double electron-electron resonance distance distributions or an X-ray structure of Cu(I)-bound Atox1. We suggest that the increased flexibility might benefit the process of ion transmission between interacting proteins. Further experiments can scrutinize this hypothesis and shed additional light on the mechanism of action of Atox1.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.

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.

The role of nucleoside triphosphate hydrolase metallochaperones in making metalloenzymes.

Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.

A role for bioinorganic chemistry in the reactivation of mutant p53 in cancer.

Metal ion dysregulation has been implicated in a number of diseases from neurodegeneration to cancer. While defective metal ion transport mechanisms are known to cause specific diseases of genetic origin, the role of metal dysregulation in many diseases has yet to be elucidated due to the complicated function (both good and bad!) of metal ions in the body. A breakdown in metal ion speciation can manifest in several ways from increased reactive oxygen species (ROS) generation to an increase in protein misfolding and aggregation. In this review, we will discuss the role of Zn in the proper function of the p53 protein in cancer. The p53 protein plays a critical role in the prevention of genome mutations via initiation of apoptosis, DNA repair, cell cycle arrest, anti-angiogenesis, and senescence pathways to avoid propagation of damaged cells. p53 is the most frequently mutated protein in cancer and almost all cancers exhibit malfunction along the p53 pathway. Thus, there has been considerable effort dedicated to restoring normal p53 expression and activity to mutant p53. This includes understanding the relative populations of the Zn-bound and Zn-free p53 in wild-type and mutant forms, and the development of metallochaperones to re-populate the Zn binding site to restore mutant p53 activity. Parallels will be made to the development of multifunctional metal binding agents for modulating the aggregation of the amyloid-beta peptide in Alzheimer's Disease (AD).

Metallochaperones: A critical regulator of metal homeostasis and beyond.

Metallochaperones are a class of unique protein families that was originally found to interact with cellular metal ions by metal delivery to specific target proteins such as metal enzymes. Recently, some members of metallochaperones receive much attention owning to their multi-biological functions in mediating plant growth, development and biotic or abiotic stress responses, particularly in the aspects of metal transport and accumulation in plants. For example, some non-essential toxic heavy metals (e.g. cadmium and mercury) accumulating in farmland due to the industrial and agronomic activities, are a constant threat to crop production, food safety and human health. Digging genetic resources and functional genes like metallochaperones is critical for understanding the metal detoxification in plants, and may help develop cleaner crops with minimal toxic metals in leafy vegetables and grains, or plants for metal-polluted soil phytoremediation. In this review, we highlight the current advancement of the research on functions of metallochaperones in metal accumulation, detoxification and homeostasis. We also summarize the recent progress of the research on the critical roles of the metal-binding proteins in regulating plant responses to some other biological processes including plant growth, development, pathogen stresses, and abiotic stresses such salt, drought, cold and light. Finally, an additional capacity of some members of metallochaperones involved in the resistance to the pathogen attack and possibly regulatory roles was reviewed.

The function of Scox in glial cells is essential for locomotive ability in Drosophila.

Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. In this study, we demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]­carbon monoxide dehydrogenase.

Nickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe4S4 cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis.

A Novel Heterozygous Missense Variant in the CIAO1 Gene in a Family with Alzheimer's Disease: The Val67Ile Variant Promotes the Interaction of CIAO1 and Amyloid-ß Protein Precursor.

Familial dementia is a rare inherited disease involving progressive impairment of memory, thinking, and behavior. We report a novel heterozygous pathogenic variant (c.199G > A, p.Val67Ile) in the CIAO1 gene that appears to be co-segregated with Alzheimer's disease in a Japanese family. Biochemical analysis of CIAO1 protein revealed that the variant increases the interaction of CIAO1 with immature amyloid-ß protein precursor (AßPP), but not mature or soluble AßPP, indicating plausible CIAO1 involvement in AßPP processing. Our study indicates that a heterozygous variant in the CIAO1 gene may be closely related to autosomal dominant familial dementia.