Property of a new Prussian blue-bounded iron complex able to peroxidate non-saturated fatty acids with a tendency to create conditions that may encourage ferroptosis.

Ferroptosis is a cell death process caused by redox imbalance in the cell environment. However, the cell death pathway proves beneficial in anticancer therapy, so compounds inducing ferroptosis are sought. The paper presents a newly synthesized iron complex named FeT, composed of ferricyanide and tartrate, which seems to meet these expectations. It is relatively stable, easily soluble in water and capable of peroxidating unsaturated fatty acids. T24 bladder cells were used as model cells. Preliminary studies demonstrated a strong inhibitory effect of this compound on cell proliferation. The cytotoxicity of FeT was assessed. Independently, it initiates caspase activity, indicating the complex cellular impact of this compound. This effect is compellingly the result of FeT penetration into the cell's interior with possible direct damage to mitochondria, thus explaining the involvement of apoptosis in cell death. At the same time, after penetrating into the cell, it causes an increase in reactive oxygen species (ROS), lipid peroxidation and a decrease in reduced glutathione, which is interpreted as to cause ferroptosis. In turn, reducing mitochondrial potential may indicate both ferroptosis and an internal pathway to apoptosis.

Two-intermediate model to characterize the structure of fast-folding proteins.

This paper introduces a new model that enables researchers to conduct protein folding simulations. A two-step in silico process is used in the course of structural analysis of a set of fast-folding proteins. The model assumes an early stage (ES) that depends solely on the backbone conformation, as described by its geometrical properties--specifically, by the V-angle between two sequential peptide bond planes (which determines the radius of curvature, also called R-radius, according to a second-degree polynomial form). The agreement between the structure under consideration and the assumed model is measured in terms of the magnitude of dispersion of both parameters with respect to idealized values. The second step, called late-stage folding (LS), is based on the "fuzzy oil drop" model, which involves an external hydrophobic force field described by a three-dimensional Gauss function. The degree of conformance between the structure under consideration and its idealized model is expressed quantitatively by means of the Kullback-Leibler entropy, which is a measure of disparity between the observed and expected hydrophobicity distributions. A set of proteins, representative of the fast-folding group - specifically, cold shock proteins - is shown to agree with the proposed model.

Why do antifreeze proteins require a solenoid?

Proteins whose presence prevents water from freezing in living organisms at temperatures below 0 °C are referred to as antifreeze proteins. This group includes molecules of varying size (from 30 to over 300 aa) and variable secondary/supersecondary conformation. Some of these proteins also contain peculiar structural motifs called solenoids. We have applied the fuzzy oil drop model in the analysis of four categories of antifreeze proteins: 1 - very small proteins, i.e. helical peptides (below 40 aa); 2 - small globular proteins (40-100 aa); 3 - large globular proteins (>100 aa) and 4 - proteins containing solenoids. The FOD model suggests a mechanism by which antifreeze proteins prevent freezing. In accordance with this theory, the presence of the protein itself produces an ordering of water molecules which counteracts the formation of ice crystals. This conclusion is supported by analysis of the ordering of hydrophobic and hydrophilic residues in antifreeze proteins, revealing significant variability - from perfect adherence to the fuzzy oil drop model through structures which lack a clearly defined hydrophobic core, all the way to linear arrangement of alternating local minima and maxima propagating along the principal axis of the solenoid (much like in amyloids). The presented model - alternative with respect to the ice docking model - explains the antifreeze properties of compounds such as saccharides and fatty acids. The fuzzy oil drop model also enables differentiation between amyloids and antifreeze proteins.

Determining protein similarity by comparing hydrophobic core structure.

Formal assessment of structural similarity is - next to protein structure prediction - arguably the most important unsolved problem in proteomics. In this paper we propose a similarity criterion based on commonalities between the proteins' hydrophobic cores. The hydrophobic core emerges as a result of conformational changes through which each residue reaches its intended position in the protein body. A quantitative criterion based on this phenomenon has been proposed in the framework of the CASP challenge. The structure of the hydrophobic core - including the placement and scope of any deviations from the idealized model - may indirectly point to areas of importance from the point of view of the protein's biological function. Our analysis focuses on an arbitrarily selected target from the CASP11 challenge. The proposed measure, while compliant with CASP criteria (70-80% correlation), involves certain adjustments which acknowledge the presence of factors other than simple spatial arrangement of solids.

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM.

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 +/- 2800 in Tris - HCl buffer and 21 000 +/- 1000 in 6 M guanidine - HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10(-3) M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 - 10(6) M-1 for the apparent association constant and 4.4 - 10(-4) mol of Ca2+ bound per g of protein for high affinity bindings sites were estimated, on the basis of data from the equilibrium dialysis. The origin and possible biological role of this protein is discussed.

Intramolecular immunological signal hypothesis revived--structural background of signalling revealed by using Congo Red as a specific tool.

Micellar structures formed by self-assembling Congo red molecules bind to proteins penetrating into function-related unstable packing areas. Here, we have used Congo red--a supramolecular protein ligand--to investigate how the intramolecular structural changes that take place in antibodies following antigen binding lead to complement activation. According to our findings, Congo red binding significantly enhances the formation of antigen-antibody complexes. As a result, even low-affinity transiently binding antibodies can participate in immune complexes in the presence of Congo red, although immune complexes formed by these antibodies fail to trigger the complement cascade. This indicates that binding of antibodies to the antigen may not, by itself, fulfill the necessary conditions to generate the signal which triggers effector activity. These findings, together with the results of molecular dynamics simulation studies, enable us to conclude that, apart from the necessary assembling of antibodies, intramolecular structural changes generated by strains which associate high- affinity bivalent antibody fitting to antigen determinants are also required to cross the complement activation threshold.

Congo red-stabilized intermediates in the lambda light chain transition from native to molten state.

Disruption of tertiary interaction makes a protein accessible to penetration by different small molecular compounds. Their interaction may stabilize the altered protein conformation. Congo red is proposed here as a stabilizer of the molten globule state and also of highly reversible intermediates in the transition from native to molten state. Human immunoglobulin lambda light chain (dimer) was used. Two protein-Congo red complexes were found after heating lambda chain in the presence of Congo red. They differed in the amount of attached dye molecules. The binding of dye was interpreted as a two-step dye penetration process involving the peripheral parts of the protein in the first step (at lower temperatures). It was concluded that the liquid crystal properties of Congo red enable it to form specific complexes with proteins, which have become accessible to penetration by ligands after global or local disruption of tertiary interaction. This dye may thus be used as a stabilizer of unfolding intermediates in the step preceding the molten globule state.

Effect of self association of bis-ANS and bis-azo dyes on protein binding.

A correlation was found between the ability of dyes (ANS, bis-ANS, Congo red, Evans blue) to form self-associated supramolecular structures in water and their tendency to form complexes with proteins. The self-association ability of dyes was measured as the resistance of a molecular sieve to their penetration. Quantitative evaluation of dye-protein interaction involved measuring the effect of dye on antibodies that agglutinate sheep red blood cells. Enhancement of agglutination by dye was assumed to represent its protein complexation ability. The results confirm that, relative to monomers, self-associated ligands also have altered protein binding properties.

Supramolecular ligands: monomer structure and protein ligation capability.

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.

The hemolytic activity of (Fab-Fc) recombinant immunoglobulins with specificity for the sheep red blood cells.

Antibody-like molecules were formed by artificial recombination of proteolytic IgG fragments (Fab' with anti-SRBC activity, and Fc) and used for studies concerning the complement fixation. Such molecules, when composed of single Fab' bound to Fc fragment appeared inactive, while species containing two Fab' fragments revealed the hemolytic activity. The results were discussed and interpreted, assuming that the interaction of Fab domains with CH2 domains in the Fc fragment is a main structural effect influencing the binding of the complement.

The use of Fab-Fc recombinant antibodies for studying the mechanism of triggering the effector activities of immunoglobulins.

Immunoglobulin species, recombined from Fab and Fc fragments with anti-red cell specificity were used as cytophilic antibodies in studies of triggering the effector activity of immunoglobulins. Recombinant immunoglobulins containing a single Fab' appeared inactive in binding to cell receptors and triggering antibody dependent cellular cytotoxicity, while those with two Fab' exhibited an activity comparable to intact antibody IgG. The experiments indicate that Fab fragment may interact with Fc and inhibit its binding to cell receptors.

The effect of azo dyes on heat aggregation of IgG.

The mechanism of IgG heat aggregation was studied using IgG aggregates complexed with azo dyes to increase their solubility and stability. Heat dependent and heat independent steps of aggregation were differentiated. On heating IgG at the dye concentration exceeding 100 times that of protein, mainly dimers are formed, as judged from ultracentrifugation and chromatographic analysis, whereas high molecular weight derivatives appear at room temperature when the protein/dye ratio is decreased. The analysis of spectral changes following either the attachment or removal of the dye from IgG aggregates implies that only a part of the dye molecules is bound firmly and directly to the protein binding sites. These dye molecules which are easily removed by adsorption to cellulose or reduced by dithionate but migrate together with IgG aggregates on chromatography and electrophoresis, are supposed to constitute that part of the micelle which extrudes from the binding site and, hence, is fixed indirectly to protein. Various proteins with predominant beta-structure were also found to bind azo dyes when heated.

Supramolecularity creates nonstandard protein ligands.

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.

Deficient polymerization of human IgM.

It has been suggested that the low disulfide interchange activity in cells producing IgM causes the excretion of immunoglobulin molecules with deficient disulfide cross-linking. IgM material found in the serum formed aggregates which were stabilized largely by noncovalent bonds. Sedimentation coefficients of these aggregates ranged from 11 to 19 S and were similar to sedimentation coefficients calculated for molecules composed of 2, 3, 4 and 5 IgM subunits. A fraction corresponding to a single IgM subunit with a sedimentation coefficient 7.1 S was also present in the serum. The molecular weight of this fraction, determined in mild conditions by Sephadex gel filtration, appeared higher than that predicted for IgMs, reaching the value of 270 000-280 000. The evident increment of molecular weight, compared to a single IgM subunit, was attributed to glycopeptides of sedimentation coefficient 2 S, which form the complex with IgMs molecules. These glycopeptides were also present in aggregates consisting of more than one IgM subunit. The number of IgM subunits participating in aggregates and their molecular weights appeared to depend on the ratio of 2 S glycopeptides to IgM material. With the increase of this ratio the molecular weight of the aggregates lowered and vice versa. Electrical charge and some physico-chemical properties of the aggregates were also significantly affected by the presence of glycopeptides, 2 S glycopeptides were supposed to be the basement membrane related material. Suggestions concerning the interpretation of the phenomenon are presented and discussed.

The use of thiosulfate to increase polymerization of IgM subunits.

Sodium thiosulfate was used to enhance in vivo the polymerization of myeloma IgM, deficient in disulfide cross-links. The therapy sharply decreased the amount of low molecular weight IgM fractions, while increasing the serum content of molecules of higher molecular weight. The degree of disulfide cross-linking in IgM increased under the influence of thiosulfate. The rate of secretion into the serum and urine of some membrane-related glycopeptides and species rich in sialic acid was reduced. Also, the discharge of L chains to the urine was lowered during the thiosulfate trial. All these changes were attributed to enhancement of disulfide-interchanging enzyme activity by thiosulfate.

Lysozyme folded in silico according to the limited conformational sub-space.

The conformational sub-space oriented on early-stage protein folding is applied to lysozyme folding. The part of the Ramachandran map distinguished on the basis of a geometrical model of the polypeptide chain limited to the mutual orientation of the peptide bond planes is shown to deliver the initial structure of the polypeptide for the energy minimization procedure in the ab initio model of protein folding prediction. Two forms of energy minimization and molecular dynamics simulation procedures were applied to the assumed early-stage protein folding of lysozyme. One of them included the disulphide bond system and the other excluded it. The post-energy-minimization and post-dynamics structures were compared using RMS-D and non-bonding contact maps to estimate the degree of approach to the native, target structure of the protein molecule obtained using the limited conformational sub-space for the early stage of folding.

The effect of azo dyes on the formation of immune complexes.

The azo dyes were found to react with antibodies aggregated in immune complex in a similar way to heat-aggregated IgG. The whole micelles of the dye, instead of single molecules, are fixed to antibodies. In a consequence, the number of dye molecules, determined per one antibody molecule, differs but may be as large as 50-60. The dye, bound to antibody complexed with antigen, enhances its affinity to antigen. The increase of affinity, expressed as the relation of changed by the dye association constants to its initial value, was found to be 6.27 x 10(8):1. The corresponding energy change, calculated from this value, equals 11.97 kcal/mol. The resulting amount of antibodies, bound to antigen, is essentially enlarged. The enhancement effect does not seem to depend on the number of antibodies bound to the red cell up to about 3 x 10(4) molecules. It indicates that in this range a single antibody binding may be affected by the dye. The situation changes at growing antibody densities in the complex when the dye-antibody network, formed by crossbridged molecules, additionally increases the stability of the immunoglobulin molecules engaged in the complex.

The formation of soluble heat IgG aggregates for immunological studies.

The structural alteration associated with the aggregation of heated immunoglobulin molecules and the triggering of their effector reactivity was studied using soluble aggregate derivatives. Large but still soluble aggregate species were obtained by the formation of complexes of heated immunoglobulin molecules with azo dyes - Congo Red or Trypan Blue. Exposed peptide fragments in the area rearranged on heating were identified by short-lasting proteolysis. The altered area was in this way localized in the Fab portion of the heavy chain. The heavy chain fragment bordered by disulphides which stabilize the domains V and CH1 was indicated as a most probable peptide portion forming the link between the aggregating molecules. The obtained dye-aggregate species retain their effector activity. The effect of structural alterations in the Fab fragment on the Fc remains unclear.

Characterization of peptides found in human biological fluids in macroglobulinemia IgM.

Excessive production of IgM in macroglobulinemia is accompanied frequently by secretion of proteins and peptides which are normally absent from biological fluids. These proteins and peptides are probably secreted by cells producing IgM. Three types of peptides were isolated and their basic properties are described. One peptide showed calcium-binding properties, and the other two peptides were bound to secreted IgM immunoglobulin.

Effect of thiosulfate on the immune response in mice, in vitro.

It was found that intravenous application of thiosulfate increased non-specifically immunoglobulin concentration in mice sera. Thiosulfate stimulated the immune response to T-dependent antigens.