Bacterial sensors define intracellular free energies for correct enzyme metalation.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

Metalloprotein switches that display chemical-dependent electron transfer in cells.

Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe-2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.

TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10.

Cancer cells adopt various modes of migration during metastasis. How the ubiquitination machinery contributes to cancer cell motility remains underexplored. Here, we report that tripartite motif (TRIM) 59 is frequently up-regulated in metastatic breast cancer, which is correlated with advanced clinical stages and reduced survival among breast cancer patients. TRIM59 knockdown (KD) promoted apoptosis and inhibited tumor growth, while TRIM59 overexpression led to the opposite effects. Importantly, we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells. At the molecular level, we identified programmed cell death protein 10 (PDCD10) as a target of TRIM59. TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 (RNFT1)-induced lysine 63 (K63) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa (p62)-selective autophagic degradation. TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A (RhoA)-Rho-associated coiled-coil kinase (ROCK) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness. PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes, thereby accelerating cell migration, invasion, and tumor formation. These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer.

Introduction: Metals in Biology: METALS AT THE HOST-PATHOGEN INTERFACE.

This seventh Metals in Biology Thematic Series deals with the metal-based interactions of mammalian hosts with pathogens. Both pathogens and hosts have complex regulatory systems for metal homeostasis. Understanding these provides strategies for fighting pathogens, either by excluding essential metals from the microbes, by delivery of excess metals to cause toxicity, or by complexing metals in microorganisms. Intervention is possible by delivery of complexing reagents or by targeting the microbial regulatory apparatus.

Copper at the Fungal Pathogen-Host Axis.

Fungal infections are responsible for millions of human deaths annually. Copper, an essential but toxic trace element, plays an important role at the host-pathogen axis during infection. In this review, we describe how the host uses either Cu compartmentalization within innate immune cells or Cu sequestration in other infected host niches such as in the brain to combat fungal infections. We explore Cu toxicity mechanisms and the Cu homeostasis machinery that fungal pathogens bring into play to succeed in establishing an infection. Finally, we address recent approaches that manipulate Cu-dependent processes at the host-pathogen axis for antifungal drug development.

Metallophosphoesterases: structural fidelity with functional promiscuity.

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi- or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function.

Thematic minireview series: metals in biology 2013.

One-half of the available protein structures contain metals, explaining their roles as essential trace elements. Metals are also critical in many aspects of nucleic acid biochemistry. This prologue briefly introduces the fifth of the Thematic Series on Metals in Biology, which began in the Journal of Biological Chemistry in 2009. The five minireviews in this 2013 series deal with the molybdenum prosthetic group (a pterin known as Moco); the biosynthesis of the "M-cluster" molybdenum prosthetic group of nitrogenase; the biosynthesis of the nickel-based metallocenter of the enzyme urease; several of the processing, transport, and medical aspects of cobalamins; and the growing roles of heme sensor proteins.

A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase.

We have investigated the final steps of complex iron-sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron-sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability. DmsA-Cys59 is a ligand to the FS0 [4Fe-4S] cluster. In the presence of trace amounts of molybdate, the Cys59Ser variant assembles normally to the cytoplasmic membrane and supports respiratory growth on DMSO, although the ground state of FS0 as determined by EPR is converted from high-spin (S=3/2) to low-spin (S=1/2). In the presence of the molybdenum antagonist tungstate, wild-type DmsABC lacks Mo-bisPGD, but is translocated via the Tat translocon and assembles on the periplasmic side of the membrane as an apoenzyme. The Cys59Ser variant cannot overcome the dual insults of amino acid substitution plus lack of Mo-bisPGD, leading to degradation of the DmsABC subunits. This indicates that the cofactor can serve as a chemical chaperone to mitigate the destabilizing effects of alteration of the FS0 cluster. These results provide insights into the role of the Mo-bisPGD-protein interaction in stabilizing the tertiary structure of DmsA during enzyme maturation.

Interaction of metal ions with neurotransmitter receptors and potential role in neurodiseases.

There is increasing evidence that toxic metals play a role in diseases of unknown etiology. Their action is often mediated by membrane proteins, and in particular neurotransmitter receptors. This brief review will describe recent findings on the direct interaction of metal ions with ionotropic γ-aminobutyric acid (GABAA) and glutamate receptors, the main inhibitory and excitatory neurotransmitter receptors in the mammalian central nervous system, respectively. Both hyper and hypo function of these receptors are involved in neurological and psychotic syndromes and modulation by metal ions is an important pharmacological issue. The focus will be on three xenobiotic metals, lead (Pb), cadmium (Cd) and nickel (Ni) that have no biological function and whose presence in living organisms is only detrimental, and two trace metals, zinc (Zn) and copper (Cu), which are essential for several enzymatic functions, but can mediate toxic actions if deregulated. Despite limited access to the brain and tight control by metalloproteins, exogenous metals interfere with receptor performances by mimicking physiological ions and occupying one or more modulatory sites on the protein. These interactions will be discussed as a potential cause of neuronal dysfunction.

Insights into metalloenzyme microenvironments: biomimetic metal complexes with a functional second coordination sphere.

Non-covalent interactions in the second coordination sphere of metalloenzymes play a significant role in determining activity and selectivity. The importance of these interactions is reflected in the increasing number of reports on model complexes with a functional second coordination sphere. These hybrid mimics were developed according to the strategies of modification with functional groups, combination with supramolecular hosts and coupling with polymers, to shed light on the role of non-covalent interactions in metalloenzyme catalysis. This review provides an overview of recent progress in this area. An introduction to native metalloenzymes with an emphasis on the second coordination sphere is also given.

Melanotransferrin: search for a function.

BACKGROUND: Melanotransferrin was discovered in the 1980s as one of the first melanoma tumour antigens. The molecule is a transferrin homologue that is found predominantly bound to the cell membrane by a glycosyl-phosphatidylinositol anchor. MTf was described as an oncofoetal antigen expressed in only small quantities in normal tissues, but in much larger amounts in neoplastic cells. Several diseases are associated with expression of melanotransferrin, including melanoma and Alzheimer's disease, although the significance of the protein to the pathogenesis of these conditions remains unclear. SCOPE OF REVIEW: In this review, we discuss the roles of melanotransferrin in physiological and pathological processes and its potential use as an immunotherapy. MAJOR CONCLUSIONS: Although the exact biological functions of melanotransferrin remain elusive, a growing number of roles have been attributed to the protein, including iron transport/metabolism, angiogenesis, proliferation, cellular migration and tumourigenesis. GENERAL SIGNIFICANCE: The high expression of melanotransferrin in several disease states, particularly malignant melanoma, remains intriguing and may have clinical significance. Further studies on the biology of this protein may provide new insights as well as potential therapeutic avenues for cancer treatment. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Prediction of survival in diffuse large B-cell lymphoma based on the expression of 2 genes reflecting tumor and microenvironment.

Several gene-expression signatures predict survival in diffuse large B-cell lymphoma (DLBCL), but the lack of practical methods for genome-scale analysis has limited translation to clinical practice. We built and validated a simple model using one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). LIM domain only 2 (LMO2) was validated as an independent predictor of survival and the "germinal center B cell-like" subtype. Expression of tumor necrosis factor receptor superfamily member 9 (TNFRSF9) from the DLBCL microenvironment was the best gene in bivariate combination with LMO2. Study of TNFRSF9 tissue expression in 95 patients with DLBCL showed expression limited to infiltrating T cells. A model integrating these 2 genes was independent of "cell-of-origin" classification, "stromal signatures," IPI, and added to the predictive power of the IPI. A composite score integrating these genes with IPI performed well in 3 independent cohorts of 545 DLBCL patients, as well as in a simple assay of routine formalin-fixed specimens from a new validation cohort of 147 patients with DLBCL. We conclude that the measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL.

MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan-derived peptides in γ-proteobacteria.

The murein peptide amidase MpaA is a cytoplasmic enzyme that processes peptides derived from the turnover of murein. We have purified the enzyme from Escherichia coli and demonstrated that it efficiently hydrolyses the γ-D-glutamyl-diaminopimelic acid bond in the murein tripeptide (L-Ala-γ-D-Glu-meso-Dap), with Km and kcat values of 0.41±0.05 mM and 38.3±10 s-1. However, it is unable to act on the murein tetrapeptide (L-Ala-γ-D-Glu-meso-Dap-D-Ala). E. coli MpaA is a homodimer containing one bound zinc ion per chain, as judged by mass spectrometric analysis and size-exclusion chromatography. To investigate the structure of MpaA we solved the crystal structure of the orthologous protein from Vibrio harveyi to 2.17 Å (1Å=0.1 nm). Vh_MpaA, which has identical enzymatic and biophysical properties to the E. coli enzyme, has high structural similarity to eukaryotic zinc carboxypeptidases. The structure confirms that MpaA is a dimeric zinc metalloprotein. Comparison of the structure of MpaA with those of other carboxypeptidases reveals additional structure that partially occludes the substrate-binding groove, perhaps explaining the narrower substrate specificity of the enzyme compared with other zinc carboxypeptidases. In γ-proteobacteria mpaA is often located adjacent to mppA which encodes a periplasmic transporter protein previously shown to bind murein tripeptide. We demonstrate that MppA can also bind murein tetrapeptide with high affinity. The genetic coupling of these genes and their related biochemical functions suggest that MpaA amidase and MppA transporter form part of a catabolic pathway for utilization of murein-derived peptides that operates in γ-proteobacteria in addition to the established murein recycling pathways.

Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a single VH domain.

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC(3)) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC(3) offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.

The role of metallobiology and amyloid-ß peptides in Alzheimer's disease.

The biggest risk factor for Alzheimer's disease is the process of ageing, but the mechanisms that lead to the manifestation of the disease remain to be elucidated. Why age triggers the disease is unclear but an emerging theme is the inability for a cell to efficiently maintain many key processes such as energy production, repair, and regenerative mechanisms. Metal ions are essential to the metabolic function of every cell. This review will explore the role and reported changes in metal ions in Alzheimer disease, particularly the brain, blood and cerebral spinal fluid, emphasizing how iron, copper and zinc may be involved through the interactions with amyloid precursor protein, the proteolytically cleaved peptide amyloid-beta (Aß), and other related metalloproteins. Finally, we explore the monomeric makeup of possible Aß dimers, what a dimeric Aß species from Alzheimer's disease brain tissue is likely to be composed of, and discuss how metals may influence Aß production and toxicity via a copper catalyzed dityrosine cross-link.

Proline 96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface.

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.

A complex linkage in the developmental pathway of endothelial and hematopoietic cells.

During normal vertebrate development, hematopoietic and endothelial cells form closely situated and interacting populations. Although the close proximity of cells to each other does not necessarily mean that they are relatives, accumulating evidence indicates that hematopoietic and endothelial cells are indeed close kin; they share common progenitors and each is able to become the other under certain circumstances. This article summarizes recent advances in the developmental relationship between hematopoietic and endothelial cells.

Extraribosomal function of metallopanstimulin-1: reducing paxillin in head and neck squamous cell carcinoma and inhibiting tumor growth.

Metallopanstimulin-1 (MPS-1) is a multifunctional ribosomal protein RPS27 that contains a zinc finger domain of the C(4) type. MPS-1 has been found to be increased in the sera of a number of different cancers, including head and neck squamous cell carcinoma (HNSCC). However, little is known about the effect of a high-level MPS-1 in regulating cancer cell behavior. In this study, we overexpressed MPS-1 protein in the HNSCC cell line UMSCC-1. We found MPS-1 distributes not only in the cytosol, but also in the nuclei. In addition, MPS-1 is secreted into the culture medium. In vitro and in vivo experiments show that growth of UMSCC-1 cells transfected with MPS-1 is dramatically inhibited. Moreover, we also found that with overexpressing MPS-1, UMSCC-1 cells were arrested on G0/G1 phase, cell proliferation rate was reduced, and tumor angiogenesis was impaired. Further gene array analysis, immunohistochemistry staining and Western blotting reveal that MPS-1 reduces paxillin mRNA and protein levels in UMSCC-1 cells both in vitro and in vivo. Together, these data indicate that when MPS-1 is overexpressed, it has an extraribosomal function as a strong inhibitor of HNSCC tumor cell growth, which may be exerted by reduced paxillin gene expression.

TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells.

The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. We reported that TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. Here, we investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. We found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis.

Characterizing the role of Zn cluster family transcription factor ZcfA in governing development in two Aspergillus species.

Filamentous fungi reproduce asexually or sexually, and the processes of asexual and sexual development are tightly regulated by a variety of transcription factors. In this study, we characterized a Zn2Cys6 transcription factor in two Aspergillus species, A. nidulans (AN5859) and A. flavus (AFLA_046870). AN5859 encodes a Zn2Cys6 transcription factor, called ZcfA. In A. nidulans, ΔzcfA mutants exhibit decreased fungal growth, a reduction in cleistothecia production, and increased asexual reproduction. Overexpression of zcfA results in increased conidial production, suggesting that ZcfA is required for proper asexual and sexual development in A. nidulans. In conidia, deletion of zcfA causes decreased trehalose levels and decreased spore viability but increased thermal sensitivity. In A. flavus, the deletion of the zcfA homolog AFLA_046870 causes increased conidial production but decreased sclerotia production; these effects are similar to those of zcfA deletion in A. nidulans development. Overall, these results demonstrate that ZcfA is essential for maintaining a balance between asexual and sexual development and that some roles of ZcfA are conserved in Aspergillus spp.