Lactoferrin modified by hypohalous acids: Partial loss in activation of human neutrophils.

Previously we have shown that lactoferrin (LTF), a protein of secondary neutrophilic granules, can be efficiently modified by hypohalous acids (HOCl and HOBr), which are produced at high concentrations during inflammation and oxidative/halogenative stress by myeloperoxidase, an enzyme of azurophilic neutrophilic granules. Here we compared the effects of recombinant human lactoferrin (rhLTF) and its halogenated derivatives (rhLTF-Cl and rhLTF-Br) on functional responses of neutrophils. Our results demonstrated that after halogenative modification, rhLTF lost its ability to induce mobilization of intracellular calcium, actin cytoskeleton reorganization, and morphological changes in human neutrophils. Moreover, both forms of the halogenated rhLTF prevented binding of N-acetylglucosamine-specific plant lectin Triticum vulgaris agglutinin (WGA) to neutrophils and, in contrast to native rhLTF, inhibited respiratory burst of neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and by two plant lectins (WGA and PHA-L). However, we observed no differences between the effects of rhLTF, rhLTF-Cl, and rhLTF-Br on respiratory burst of neutrophils induced by phorbol 12-myristate 13-acetate (PMA), digitonin, and number of plant lectins with different glycan-binding specificity. Furthermore, all rhLTF forms interfered with PMA- and ionomycin-induced formation of neutrophil extracellular traps. Thus, halogenative modification of LTF is one of the mechanisms involved in modulating a variety of signaling pathways in neutrophils to control their pro-inflammatory activity.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein.

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.

Interaction of macrophage migration inhibitory factor with ceruloplasmin: role of labile copper ions.

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is a target for pharmacological treatment of sepsis and malignant tumors. Inhibition of tautomerase activity of MIF in reaction with p-hydroxyphenylpyruvate (HPP) was observed in the presence of ceruloplasmin (CP), a copper-containing plasma protein. Binding labile copper ions to CP (CP+Cu(II)) is a prerequisite for MIF inhibiting. CP+Cu(II) is shown to be an uncompetitive inhibitor of MIF (Ki ~ 37 nM), which suggests formation of a complex 'MIF-HPP-CP-Cu(II)'. Filtration of CP+Cu(II) on a column with Chelex-100, otherwise the presence of high concentrations of histidine, cysteine or methionine abrogated the inhibitory effect of CP. Adding salts of Co(II) and Ni(II) that replace copper ions in the labile sites prevented the inhibitory effect of CP+Cu(II). Limited proteolysis of CP by thrombin diminished its oxidase activity in reaction with p-phenylenediamine, but endowed it with the capacity of inhibiting MIF. Covalent modification of MIF by phenylmethylsulfonyl fluoride (PMSF) resulted in binding of MIF-PMSF to CP immobilized on CM5 chip, the dissociation constant being 4.2 µM. In D-galactosamine-sensitized mice CP+Cu(II) increased the LPS-induced lethality from 54 to 100%, while administration of antibodies against MIF prevented the lethal effect. The enhancement by CP+Cu(II) of the pro-inflammatory signal of MIF is discussed.

A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis.

The fibrillogenesis of a peptide corresponding to residues 35-51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹7) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.

Myeloperoxidase modulates human platelet aggregation via actin cytoskeleton reorganization and store-operated calcium entry.

Myeloperoxidase (MPO) is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist). In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca(2+) through enhancement of store-operated Ca(2+) entry (SOCE). Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.

Amyloidogenic peptide homologous to fragment 129-148 of human myocilin.

Myocilin is a protein with a molecular weight near 50 kDa. It is expressed in almost all organs and tissues. We showed that the peptide DQLETQTRELETAYSNLLRD corresponding to N-terminal Leucine zipper motif (LZM) of the protein is able to form amyloid-like fibrils. The possible role of this motif in myocilin aggregation is discussed.