The role of zinc in the S100 proteins: insights from the X-ray structures.

We here aim to summarise the present knowledge on zinc binding by S100 proteins. While the importance of modulation of the function of the S100 family of EF-hand proteins by calcium is well established, a substantial proportion is also regulated by zinc or copper. Indeed regulation by zinc in addition to calcium was suggested almost as soon as the first S100 protein was discovered and has been confirmed for many family members by numerous experiments. For the first, "His-Zn", group, zinc-binding sites composed of three histidines and an aspartic acid were first proposed based on sequence comparisons and later confirmed by structural studies. A second, "Cys-Zn", group lacks such well-defined zinc-binding motifs and for these cysteines were suggested as the main zinc ligands. There is no three-dimensional structure for a Cys-Zn S100 in the presence of zinc. However, analysis of their sequences together with their X-ray structures in the absence of zinc suggests the possibility of two zinc-binding sites: a conserved site with a degree of similarity to those of the His-Zn group and a less-defined site with a Cys interdimer-binding motif. Some S100 protein-mediated events, such as signalling in the extracellular space, where the levels of calcium are already high, are most unlikely to be calcium regulated. Therefore, a broader knowledge of the role of zinc in the functioning of the S100 proteins will add significantly to the understanding how they propagate their signals.

Effect of enzymatic deimination on the conformation of recombinant prion protein.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function.

BACKGROUND: Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. RESULTS: The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE--the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. CONCLUSION: We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

Proteolysis of prion protein by cathepsin S generates a soluble beta-structured intermediate oligomeric form, with potential implications for neurotoxic mechanisms.

Formation of PrP aggregates is considered to be a characteristic event in the pathogenesis of TSE diseases, accompanied by brain inflammation and neurodegeneration. Factors identified as contributing to aggregate formation are of interest as potential therapeutic targets. We report that in vitro proteolysis of ovine PrP(94-233) (at neutral pH and in the absence of denaturants) by the protease cathepsin S, a cellular enzyme that also shows enhanced expression in pathogenic conditions, occurs selectively in the region 135-156. This results in an unusually efficient, concentration-dependent conformational conversion of a large subfragment of PrP(94-233) into a soluble beta-structured oligomeric intermediate species, that readily forms a thioflavin-T-positive aggregate. N-terminal sequencing of the proteolysis fragments shows the aggregating species have marked sequence similarities to truncated PrP variants known to confer unusually severe pathogenicity when transgenically expressed in PrP(o/o) mice. Circular dichroism analysis shows that PrP fragments 138-233, 144-233 and 156-233 are significantly less stable than PrP(94-233). This implies an important structural contribution of the beta1 sequence within the globular domain of PrP. We propose that the removal or detachment of the beta1 sequence enhances beta-oligomer formation from the globular domain, leading to aggregation. The cellular implications are that specific proteases may have an important role in the generation of membrane-bound, potentially toxic, beta-oligomeric PrP species in pre-amyloid states of prion diseases. Such species may induce cell death by lysis, and also contribute to the transport of PrP to neuronal targets with subsequent amplification of pathogenic effects.

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro.

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.

Thiolated PAMAM dendrimer-coated CdSe/ZnSe nanoparticles as protein transfection agents.

Functionalisation of PAMAM dendrimers with a small number of thiol groups makes them good ligands for CdSe/ZnSe nanoparticles; the particles coated with thiolated dendrimers have good cell permeability and are potent transfection agents.

Human DNA topoisomerase I: An anticancer drug target present in human sarcomas.

New anticancer drugs targeting DNA topoisomerase I (topo I) are showing activity against human sarcomas. Laboratory studies have indicated that cells responsive to topo I-targeted drugs have elevated levels of topo I, require active DNA replication, and may require a functional apoptotic pathway. In this study, we evaluated these potential markers of topo I-targeted drug sensitivity in 55 cases of human sarcoma (42 high grade, 4 intermediate grade, and 9 low grade). By immunohistochemical staining, we observed elevated topo I expression in 20 of 55 neoplasms (36%). Immunohistochemical staining for the proliferation marker DNA topoisomerase II-alpha (topo II-alpha), showed that 15 of 55 neoplasms (27%) had topo II-alpha indices >50, indicating a large number of actively cycling tumor cells. Abnormal p53 expression was observed in 19 of the 55 cases (35%). None of the cases were interpreted as positive for ALK-1. To complement our immunohistochemical staining of topo I, we isolated functionally active topo I from extracts of a human sarcoma. These isolates demonstrated that sarcoma topo I is sensitive to topo I-targeted anticancer drugs. Of the 55 cases of human sarcoma, 7 (13%) had high levels of topo I, a large number of cycling tumor cells, and normal p53 expression. These are the molecular parameters that might suggest responsiveness to drugs targeting topo I.

Implications of protein structure instability: from physiological to pathological secondary structure.

Proteins are folded during their synthesis; this process may be spontaneous or assisted. Both phenomena are carefully regulated by the "housekeeping" mechanism and molecular chaperones to avoid the appearance of misfolded proteins. Unfolding process generally occurs during physiological degradation of protein, but in some specific cases it results from genetic or environmental changes and does not correspond to metabolic needs. The main outcome of these phenomena is the appearance of nonfunctional pathologically unfolded proteins with a strong tendency to aggregation. Moreover, for some of these unfolded proteins, the agglomeration that follows initial proteins association may give rise to highly structured soluble aggregates. These aggregates have been identified as the main cause of the so-called amyloidosis or amyloid diseases, such as Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases, and type II diabetes mellitus. Although some common mechanisms of amyloid protein aggregation have been identified, the roles of the environmental conditions inducing amyloidosis remain to be clarified. In this review, we will summarize recent studies identifying the origin of amyloid nucleation and will try to predict the therapeutic prospects that may be opened by elucidation of the amyloidosis mechanisms.

The crystal structure of zebrafish S100Z: implications for calcium-promoted S100 protein oligomerisation.

The S100 family, with about 20 members in humans, is composed of EF-hand calcium-regulated proteins and is linked to a range of serious human diseases, including cancer and autoimmune and neurological disorders. The oldest S100 family members are found in teleosts (bony fish). The zebrafish, Danio rerio, was suggested as a promising model system for in vivo studies on S100 family functions, and we chose to investigate zebrafish S100Z as the closest homologue of the metastasis-promoting human S100A4. Here, we report the first crystal structure of an S100 protein from this organism, the calcium-bound state of S100Z to 2.03 Å resolution. Crystal packing suggests higher-order oligomerisation of S100Z dimers, with a tetramerisation interface very similar to, but even more extensive than, that reported for S100A4. The interactions are primarily through the C-terminal αIV helices from adjacent dimers in an antiparallel orientation. Structural comparisons between known S100 multimeric assemblies together with analysis of calcium-driven changes to the dimerisation cores suggest a mechanism for calcium-promoted oligomerisation of S100 proteins.

The crystal structures of human S100A12 in apo form and in complex with zinc: new insights into S100A12 oligomerisation.

The functions of the members of the S100 family of EF-hand proteins are modulated by calcium and, in a number of cases, by zinc or copper. One such protein is S100A12, which is implicated in inflammation and host-parasite responses. Previously, we reported the structures of human S100A12 in both low (dimeric) and high (hexameric) calcium forms and, in addition, that of a complex with copper and calcium. Here we report the crystal structures of the metal-free apo form of human S100A12 at 1.77 A resolution and of the zinc complex in two crystal forms (P2(1)2(1)2(1) and F222) to 1.88 A and 1.73 A resolution, respectively. These are the first structures of a zinc-only complex of an S100 protein to be determined. The zinc complex structure shows significant differences from those of both calcium-loaded and apo-S100A12 structures, and comparisons suggest an explanation for the zinc-induced 1500-fold increase in calcium affinity. In addition, the new structures provide insight into the role of zinc-calcium interplay in the transition of S100A12 from a dimer through a tetramer to a hexamer. The role of both zinc and calcium in target binding by S100A12 during host-parasite responses is confirmed by experiments with paramyosin from the tropical parasites Onchocerca volvulus and Brugia malayi.

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons.

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.

Hexameric calgranulin C (S100A12) binds to the receptor for advanced glycated end products (RAGE) using symmetric hydrophobic target-binding patches.

Calgranulin C (S100A12) is a member of the S100 family of proteins that undergoes a conformational change upon calcium binding allowing them to interact with target molecules and initiate biological responses; one such target is the receptor for advanced glycation products (RAGE). The RAGE-calgranulin C interaction mediates a pro-inflammatory response to cellular stress and can contribute to the pathogenesis of inflammatory lesions. The soluble extracellular part of RAGE (sRAGE) was shown to decrease the inflammation response possibly by scavenging RAGE-activating ligands. Here, by using high resolution NMR spectroscopy, we identified the sRAGE-calgranulin C interaction surface. Ca2+ binding creates two symmetric hydrophobic surfaces on Ca2+-calgranulin C that allow calgranulin C to bind to the C-type immunoglobulin domain of RAGE. Apo-calgranulin C also binds to sRAGE using a completely different surface and with substantially lower affinity, thus underscoring the role of Ca2+ binding to S100 proteins as a molecular switch. By using native gel electrophoresis, chromatography, and fluorescence spectroscopy, we established that sRAGE forms tetramers that bind to hexamers of Ca2+-calgranulin C. This arrangement creates a large platform for effectively transmitting RAGE-dependent signals from extracellular S100 proteins to the cytoplasmic signaling complexes.

Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition.

Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.

Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons.

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.

Probing the conformation of the prion protein within a single amyloid fibril using a novel immunoconformational assay.

The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159-174 and 224-230 were shown to be buried and were the most resistant to denaturation. The 132-156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95-105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89-112 and 136-158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.

DNA binding induces conformational transition within human DNA topoisomerase I in solution.

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.