Sodium Ions as Regulators of Transcription in Mammalian Cells.

The maintenance of an uneven distribution of Na+ and K+ ions between the cytoplasm and extracellular medium is the basis for the functioning of any animal cell. Changes in the intracellular ratio of these cations occur in response to numerous stimuli and are important for the cell activity regulation. Numerous experimental data have shown that gene transcription in mammalian cells can be regulated by changes in the intracellular [Na+]i/[K+]i ratio. Here, we discuss possible mechanisms of such regulation in various cell types, with special attention to the [Ca2+]-independent signaling pathways that suggest the presence of an intracellular sensor of monovalent cations. As such sensor, we propose the secondary structures of nucleic acids called G-quadruplexes. They are widely represented in mammalian genomes and are often found in the promoters of genes encoding transcription factors.

Increased Extracellular Sodium Concentration as a Factor Regulating Gene Expression in Endothelium.

Hyperosmotic stimulation of endothelial cells often leads to its dysfunction accompanied, among other things, by proinflammatory response. The mechanisms of this phenomenon are not fully understood. It may arise due to increase in the plasma Na+ concentration, due to increase in the extracellular osmolarity, increase in the intracellular Na+i/K+i ratio, and/or change in the cell stiffness. In the present study we investigated the effects of short-term increase in osmolarity of extracellular medium on the mRNA content of some genes important for endothelial function (including Na+i/K+i-sensitive ones) and the equivalent elasticity constant of human umbilical vein endothelial cells membranes. Hyperosmotic stimulation of these cells with NaCl but not mannitol resulted in accumulation of Na+ ions inside the cells despite the Na,K-ATPase activation, and was also accompanied by the decrease in their equivalent elasticity constant. The amount of IL1α mRNA decreased with increasing osmolarity of the extracellular medium, whereas the amount of ATF3, PAR2, and PTGS2 mRNAs increased only in response to the increasing NaCl concentration. At the same time, under the conditions of our experiments, we did not detect changes in the expression of the osmoprotective transcription factor NFAT5. The obtained data indicate that the increase of extracellular Na+ concentration in the physiological range is an independent factor that affects intracellular Na+i/K+i ratio and regulates expression of some genes (in particular, ATF3, PAR2, PTGS2) in endothelial cells.

Cardio-Vascular Heat Shock Protein (cvHsp, HspB7), an Unusual Representative of Small Heat Shock Protein Family.

HspB7 is one of ten human small heat shock proteins. This protein is expressed only in insulin-dependent tissues (heart, skeletal muscle, and fat tissue), and expression of HspB7 is regulated by many different factors. Single nucleotide polymorphism is characteristic for the HspB7 gene and this polymorphism correlates with cardio-vascular diseases and obesity. HspB7 has an unusual N-terminal sequence, a conservative α-crystallin domain, and very short C-terminal domain lacking conservative IPV tripeptide involved in a small heat shock proteins oligomer formation. Nevertheless, in the isolated state HspB7 forms both small oligomers (probably dimers) and very large oligomers (aggregates). HspB7 is ineffective in suppression of amorphous aggregation of model proteins induced by heating or reduction of disulfide bonds, however it is very effective in prevention of aggregation of huntingtin fragments enriched with Gln residues. HspB7 can be an effective sensor of electrophilic agents. This protein interacts with the contractile and cytoskeleton proteins (filamin C, titin, and actin) and participates in protection of the contractile apparatus and cytoskeleton from different adverse conditions. HspB7 possesses tumor suppressive activity. Further investigations are required to understand molecular mechanisms of HspB7 participation in numerous biological processes.

The pivotal role of the beta 7 strand in the intersubunit contacts of different human small heat shock proteins.

Human alpha B-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of beta 7 strand of the alpha-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wild-type counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the beta 7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of alpha B-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the beta 7 strand participates in the intersubunit interaction of four human small heat shock proteins (alpha B-crystallin, HspB1, HspB6, HspB8) having different structure of beta2 strand of alpha-crystallin domain and different length and composition of variable N- and C-terminal tails.

Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20).

Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100-150 and 250-300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.

Effect of mutations in the beta5-beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11).

The human genome encodes ten different small heat shock proteins, each of which contains the so-called alpha-crystallin domain consisting of 80-100 residues and located in the C-terminal part of the molecule. The alpha-crystallin domain consists of six or seven beta-strands connected by different size loops and combined in two beta-sheets. Mutations in the loop connecting the beta5 and beta7 strands and conservative residues of beta7 in alphaA-, alphaB-crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the beta5-beta7 loop and in the beta7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases.

Small heat shock protein with apparent molecular mass 20 kDa (Hsp20, HspB6) is not a genuine actin-binding protein.

The interaction of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20, HspB6) with actin was investigated. Wild type Hsp20 and its S16D mutant mimicking phosphorylation of Hsp20 by cyclic nucleotide-dependent protein kinases do not affect the rate and extent of actin polymerization. Ultracentrifugation of the mixture of Hsp20 (or its S16D mutant) with isolated F-actin or F-actin containing tropomyosin, calponin or alpha-actinin resulted in co-sedimentation of less than 0.04 mol of Hsp20 monomer per mol of actin. Myofibrils of skeletal, cardiac or smooth muscle bound less than 0.04 mol of Hsp20 monomer per mol of actin and this stoichiometry was independent of phosphorylation or mutation of Ser16 of Hsp20. Since Hsp20 is not a genuine actin-binding protein, the earlier described correlation between Hsp20 phosphorylation and smooth muscle relaxation cannot be explained by direct interaction of Hsp20 with actin.

Some properties of human small heat shock protein Hsp20 (HspB6).

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa. At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial alpha-crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein. At pH 6.0, both alpha-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, alpha-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase. The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperature-dependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.

Structure and properties of avian small heat shock protein with molecular weight 25 kDa.

The primary structure of chicken small heat shock protein (sHsp) with apparent molecular weight 25 kDa was refined and it was shown that this protein has conservative primary structure 74RALSRQLSSG(83) at Ser77 and Ser81, which are potential sites of phosphorylation. Recombinant wild-type chicken Hsp25, its three mutants, 1D (S15D), 2D (S77D+S81D) and 3D (S15D+S77D+S81D), as well as delR mutant with the primary structure 74RALS-ELSSG(82) at potential sites of phosphorylation were expressed and purified. It has been shown that the avian tissues contain three forms of Hsp25 having pI values similar to that of the wild-type protein, 1D and 2D mutants that presumably correspond to nonphosphorylated, mono- and di-phosphorylated forms of Hsp25. Recombinant wild-type protein, its 1D mutant and Hsp25, isolated from chicken gizzard, form stable high molecular weight oligomeric complexes. The delR, 2D and 3D mutants tend to dissociate and exist in the form of a mixture of high and low molecular weight oligomers. Point mutations mimicking phoshorylation decrease chaperone activity of Hsp25 measured by reduction of dithiothreitol induced aggregation of alpha-lactalbumin, but increase the chaperone activity of Hsp25 measured by heat induced aggregation of alcohol dehydrogenase. It is concluded that avian Hsp25 has a more stable quaternary structure than its mammalian counterparts and mutations mimicking phosphorylation differently affect chaperone activity of avian Hsp25, depending on the nature of target protein and the way of denaturing.