Igor B. Bronstein

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 β-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 β-oligomers was substantially reduced when expression of endogenous PrPC was down-regulated by small interfering RNAs. In opposition to the β-oligomer and amyloid fibrils of rPrP, the monomeric α-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble β-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrPC is required for mediating the toxicity of abnormally folded external PrP aggregates.

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.

Structure of the intact transactivation domain of the human papillomavirus E2 protein.

Papillomaviruses cause warts and proliferative lesions in skin and other epithelia. In a minority of papillomavirus types (‘high risk’, including human papillomaviruses 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours. The papillomavirus E2 protein controls primary transcription and replication of the viral genome. Both activities are governed by a ∼200 amino-acid amino-terminal module (E2NT) which is connected to a DNA-binding carboxy-terminal module by a flexible linker. Here we describe the crystal structure of the complete E2NT module from human papillomavirus 16. The E2NT module forms a dimer both in the crystal and in solution. Amino acids that are necessary for transactivation are located at the dimer interface, indicating that the dimer structure may be important in the interactions of E2NT with viral and cellular transcription factors. We propose that dimer formation may contribute to the stabilization of DNA loops which may serve to relocate distal DNA-binding transcription factors to the site of human papillomavirus transcription initiation.

Structure of the human S100A12–copper complex: implications for host-parasite defence

S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.

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

BackgroundHuman 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.ResultsThe 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.ConclusionWe 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.

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.

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.

Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system.

S100A4 (Mts1) is a Ca2+‐binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two‐hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non‐covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys76 and/or Cys81 of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.

Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin.

A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C-terminal fragment of the myosin heavy chain inhibits phosphorylation of the myosin heavy chain by protein kinase CK2 in vitro. Mts1 might also bind directly the beta subunit of protein kinase CK2, thereby modifying the enzyme activity. Our results indicate that myosin oligomers were disassembled in the presence of Mts1. The short C-terminal fragment of the myosin heavy chain was totally soluble in the presence of an equimolar amount of Mts1 at low ionic conditions (50 mM NaCl). Depolymerization was found to be calcium-dependent and could be blocked by EGTA. Our data suggest that Mts1 can increase myosin solubility and therefore suppress its assembly.

The three-dimensional structure of human S100A12

The crystal structure of human EF-hand calcium-binding protein S100A12 in its calcium-bound form has been determined to 1.95 A resolution by molecular replacement using the structure of the S100B protein. The S100 family members are homologous to calmodulin and other related EF-hand calcium-binding proteins. Like the majority of S100 proteins, S100A12 is a dimer, with the interface between the two subunits being composed mostly of hydrophobic residues. The fold of S100A12 is similar to the other known crystal and solution structures of S100 proteins, except for the linker region between the two EF-hand motifs. Sequence and structure comparison between members of the S100 family suggests that the target-binding region in S100A12 is formed by the linker region and C-terminal residues of one subunit and the N-terminal residues of another subunit of the dimer. The N-terminal region of the target-binding site includes two glutamates that are conserved in most of the S100 sequences. The comparison also provided a better understanding of the role of the residues important for intra- and inter-subunit hydrophobic interactions. The precise role of S100A12 in cell behaviour is yet undefined, as is the case for the whole family, although it has been shown that the interaction of S100A12 with the RAGE receptor is implicated in inflammatory response.