Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription.

The HSPs (heat-shock proteins) of the 70-kDa family, the constitutively expressed HSC70 (cognate 70-kDa heat-shock protein) and the stress-inducible HSP70 (stress-inducible 70-kDa heat-shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK-21) cells released HSP70 and HSC70 in a serum-free medium and that this process was the result of an active secretion of HSPs rather than the non-specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non-classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl-beta-cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK-21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK-21 cells in a serum-free medium through a non-classical pathway in which lipid rafts play an important role.

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.

Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections.

The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase-and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase-and chloratetreated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membraneassociated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.