Some properties of human small heat shock protein Hsp22 (H11 or HspB8)

Untagged recombinant human small heat shock protein with apparent molecular mass 22 kDa (Hsp22) was obtained in homogeneous state. Size exclusion chromatography and chemical crosslinking with dimethylsuberimidate indicate that Hsp22 forms stable dimers. Being highly susceptible to oxidation Hsp22 forms disulfide crosslinked dimers and poorly soluble high molecular mass oligomers. According to CD spectroscopy oxidation of Hsp22 results in disturbing of both secondary and tertiary structure. Hsp22 possesses a negligibly low autophosphorylation activity and under the conditions used is unable to phosphorylate casein or histone. Hsp22 effectively prevents heat-induced aggregation of yeast alcohol dehydrogenase and bovine liver rhodanese with chaperone activity comparable to that of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20).     

The problem of protein kinase activity of small heat shock protein Hsp22 (H11 or HspB8)

The recently described protein denoted H11, Hsp22 or HspB8 seems to participate in regulation of proliferation, apoptosis, and cardiac hypertrophy. Mutation of Hsp22 causes distal motor neuropathy. Multitude action of Hsp22 is supposed to be due to its protein kinase and/or chaperone-like activities. There are many indirect evidences indicating that Hsp22 possesses intrinsic protein kinase activity. However, low homology to protein kinases, low extent of autophosphorylation, lack of significant protein kinase activity with commonly used substrates, and lack of information on stoichiometry, kinetics, and substrate specificity make the existence of intrinsic protein kinase activity of Hsp22 questionable. It is supposed that protein kinase activity ascribed to Hsp22 is due to contaminating protein kinases. Hsp22 is highly homologous to small heat shock proteins and effectively prevents aggregation of denatured protein both in vitro and in vivo. Therefore, it is supposed that chaperone-like activity is of great importance for Hsp22 functioning.     

Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells

Estrogen (E2) plays a critical role in the etiology and progression of human breast cancer. The estrogenic response is complex and not completely understood, including in terms of the involved responsive genes. Here we show that Hsp22 (synonyms: HspB8, E2lG1, H11), a member of the small heat shock protein (sHSP) superfamily, was induced by E2 in estrogen receptor-positive MCF-7 breast cancer cells, resulting in an elevated Hsp22 protein level, whereas it was not induced in estrogen receptor-negative MDA-MB-231 cells. This induction was prevented by the pure anti-estrogen ICI182780 (faslodex, fulvestrant), whereas tamoxifen, a substance with mixed estrogenic and antiestrogenic properties, had no major inhibitory effect on this induction, nor did it induce Hsp22 on its own. Cadmium (Cd) is an environmental pollutant with estrogenic properties (metalloestrogen) that has been implicated in breast cancer. Treatment of MCF-7 cells with Cd also resulted in induction of Hsp22, and this induction was also inhibited by ICI182780. In live MCF-7 cells, Hsp22 interacted at the level of dimers with Hsp27, a related sHSP, as was shown by quantitative fluorescence resonance energy transfer measurements. In cytosolic extracts of MCF-7 cells, most of the E2- and Cd-induced Hsp22 was incorporated into high-molecular mass complexes. In part, Hsp22 and Hsp27 were components of distinct populations of these complexes. Finally, candidate elements in the Hsp22 promoter were identified by sequence analysis that could account for the induction of Hsp22 by E2 and Cd. Taken together, Hsp22 induction represents a new aspect of the estrogenic response with potential significance for the biology of estrogen receptor-positive breast cancer cells.     

Biochemical characterization of small heat shock protein HspB8 (Hsp22)–Bag3 interaction

Interaction of human Bag3 with small heat shock proteins HspB6, HspB8 and its K141E mutant was analyzed by different biochemical methods. The data of size-exclusion chromatography indicate that the wild type HspB8 forms tight complexes with Bag3. K141E mutant of HspB8 and especially HspB6 weaker interact with Bag3. The data of chemical crosslinking and analytical ultracentrifugation indicate that in vitro the stoichiometry of complexes formed by HspB8 and Bag3 is variable and is dependent on concentration of protein partners. Interaction of Bag3 and HspB8 is accompanied by increase of thermal stability measured by intrinsic tryptophan fluorescence and increased resistance to limited chymotrypsinolysis. The data of size-exclusion chromatography, analytical ultracentrifugation and limited proteolysis indicate that Bag3 belongs to the group of intrinsically disordered proteins. It is supposed that having unordered structure Bag3 might weakly interact with different small heat shock proteins which recognize unfolded proteins and this interaction is especially strong with intrinsically disordered HspB8. The complexes formed by Bag3 and HspB8 might have variable stoichiometry and can participate in different processes including clearing of the cell from improperly folded proteins.     

Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase

A number of phosphomimicking mutants (replacement of Ser/Thr residues by Asp) of human small heat shock protein HspB8 were obtained and phosphorylation of the wild type HspB8 and its mutants by ERK1 kinase was analyzed in vitro. Mutation S159D does not affect phosphorylation, whereas mutations S24D and S27D equally moderately inhibited and mutation T87D strongly inhibited phosphorylation of HspB8. The double mutations S24D/T87D and S27D/T87D induced very strong inhibitory effect and the triple mutations S24D/S27D/T87D completely prevented phosphorylation catalyzed by ERK1. Thus, Ser24 and Thr87, found to be phosphorylated in vivo, are among the sites phosphorylated by ERK1 in HspB8 in vitro. Mutations S24D and T87D affect intrinsic tryptophan fluorescence and susceptibility to chymotrypsinolysis of HspB8. Phosphomimicking mutations and phosphorylation promote concentration-dependent association of HspB8 subunits. Mutations S24D and S27D decrease, whereas mutation T87D increases the chaperone-like activity of HspB8. It is concluded that phosphorylation catalyzed by ERK1 might affect the structure and chaperone-like activity of HspB8 and therefore can be important for regulation of interaction of HspB8 with different target proteins.     

siRNA-mediated knockdown of SMC1A expression suppresses the proliferation of glioblastoma cells

SMC1A is a member of cohesin complex which has essential functions in cell cycle progression and DNA repair. Therefore, we choose SMC1A as a target gene therapy of glioblastoma. It is well known that glioblastoma has very low survival rate because of ineffectiveness of conventional treatments. This study was designed to explore the possibilities of small interfering RNA (siRNA)-mediated SMC1A silencing as alternative method of treatment. We found that the lentivirus-mediated RNAi system efficiently decreased the expression level of SMC1A. Inhibiting SMC1A expression efficiently (P < 0.001) resulted in inhibiting the proliferation and colony formation of U251 and U87MG cells. Moreover, we found that SMC1A silencing led to S cell-cycle arresting. Collectively, these results demonstrated the possibility of siRNA-mediated silencing of SMC1A as a therapeutic tool for the treatment of glioblastoma.     

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.     

The chaperone-like activity of rat HspB8/Hsp22 and dynamic molecular transition related to oligomeric architectures in vitro

HspB8/Hsp22 is a functionally distinct small heat shock proteins (sHsp) and is preferentially expressed in brain, heart, skeletal, and smooth muscle. HspB8 is also associated with neuromuscular function and protein quality control by proteasomes in cardiac hypertrophy. However, the molecular properties in vitro and molecular oligomerization remain uncertain. In this investigation, the rat HspB8 gene was expressed in E.coli cells, and mature HspB8 protein was efficiently prepared. The chaperone-like activity of HspB8 in vitro was quantitatively analyzed by model substrates. Size exclusion chromatography revealed that HspB8 had polydisperse oligomers and underwent dynamic molecular transition in solution, existing in a dynamic equilibrium between various oligomers. In a nonphysiological solution, HspB8 was predominantly octamers. In a physiological solution (pH 7.4), HspB8 mainly formed tetramers. The dynamic interactive transition maybe was helpful to maintain its molecular complxes in solution. In a FRET assay, subunit exchange occurred frequently between the various oligomers with a rate of 0.12, 0.089, and 0.064 min(-1) at 50°C, 43°C, and 37°C, respectively. It also demonstrated the dynamic molecular properties of HspB8 in solution.     

Cardiac H11 kinase/Hsp22 stimulates oxidative phosphorylation and modulates mitochondrial reactive oxygen species production: Involvement of a nitric oxide-dependent mechanism

H11 kinase/Hsp22 (Hsp22), a small heat shock protein upregulated by ischemia/reperfusion, provides cardioprotection equal to ischemic preconditioning (IPC) through a nitric oxide (NO)-dependent mechanism. A main target of NO-mediated preconditioning is the mitochondria, where NO reduces O₂ consumption and reactive oxygen species (ROS) production during ischemia. Therefore, we tested the hypothesis that Hsp22 overexpression modulates mitochondrial function through an NO-sensitive mechanism. In cardiac mitochondria isolated from transgenic (TG) mice with cardiac-specific overexpression of Hsp22, mitochondrial basal, ADP-dependent, and uncoupled O₂ consumption was increased in the presence of either glucidic or lipidic substrates. This was associated with a decrease in the maximal capabilities of complexes I and III to generate superoxide anion in combination with an inhibition of superoxide anion production by the reverse electron flow. NO synthase expression and NO production were increased in mitochondria from TG mice. Hsp22-induced increase in O₂ consumption was abolished either by pretreatment of TG mice with the NO synthase inhibitor l-N^G-nitroarginine methyl ester (l-NAME) or in isolated mitochondria by the NO scavenger phenyltetramethylimidazoline-1-oxyl-3-oxide. l-NAME pretreatment also restored the reverse electron flow. After anoxia, mitochondria from TG mice showed a reduction in both oxidative phosphorylation and H₂O₂ production, an effect partially reversed by l-NAME. Taken together, these results demonstrate that Hsp22 overexpression increases the capacity of mitochondria to produce NO, which stimulates oxidative phosphorylation in normoxia and decreases oxidative phosphorylation and reactive oxygen species production after anoxia. Such characteristics replicate those conferred by IPC, thereby placing Hsp22 as a potential tool for prophylactic protection of mitochondrial function during ischemia.     

Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders

Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.     

Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.     

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.     

Localization of human c-mos to chromosome band 8q11 in leukemic cells with the t(8;21) (q22;q22)

Summary Chromosome in situ hybridization studies locate c-mos to chromosome band 8q11 in leukemic cells carrying the t(8;21) (q22;q22). This amends the previous assignment of c-mos to chromosome band 8q22 and conforms with its recent assignment to 8q11 in normal cells and in a cell line with a structurally abnormal chromosome 8. C-mos lies proximally to, and distant from, the breakpoint at 8q22 in the t(8;21) and is unlikely to have a role in the onset of acute myeloid leukemia characterized by this translocation.     

Conditional knockdown of proteasomes results in cell-cycle arrest and enhanced expression of molecular chaperones Hsp70 and Hsp40 in chicken DT40 cells

The 26 S proteasome is an evolutionarily conserved ATP-dependent protease complex that degrades poly-ubiquitinated proteins and plays essential roles in a critical part of cellular regulation. In vertebrates, the roles of the proteasome have been widely studied by use of specific inhibitors, but not genetically. Here, we generated a cell line Z(-/-/-)/Z-HA, in which the expression of the catalytic subunit of the proteasome, Z (beta2) could be manipulated. This cell line expresses exogenous Z protein under the control of a tetracycline-repressible promoter in a Z-nullizygous genetic background. Treatment of these cells with doxycycline inhibited Z expression and, hence, the function of the proteasome. The latter resulted in accumulation of poly-ubiquitinated proteins and concomitant induction of molecular chaperones Hsp70 and Hsp40. These results suggest a synergistic role for the proteasome with these molecular chaperones to eliminate misfolded or damaged proteins in vivo. Furthermore, knockdown of the proteasome induced apoptotic cell death following cell-cycle arrest at G(2)/M phase. Our Z(-/-/-)/Z-HA cell line would be useful for evaluating proteolytic processes catalyzed by the proteasome in many biological events in vertebrate cells.     

Rosmarinic acid and siRNA combined therapy represses Hsp27 (HSPB1) expression and induces apoptosis in human glioma cells

High expression of Hsp27 in glioma cells has been closely associated with tumor cell proliferation and apoptosis inhibition. The aim of the present study was to asses the effects of rosmarinic acid (RA) on Hsp27 expression and apoptosis in non-transfected and transfected human U-87 MG cells. The effect of rosmarinic acid was compared to quercetin, which is known to be a good Hsp27 inhibitor. In order to block the expression of Hsp27 gene (HSPB1), transfection with specific siRNAs was performed. Western blotting technique was used to assess the Hsp27 expression, and caspase-3 colorimetric activity assay was performed to determine apoptosis induction. According to the results, it was found that RA and quercetin effectively silenced Hsp27 and both agents induced apoptosis by activating the caspase-3 pathway. Eighty and 215 μM RA decreased the level of Hsp27 by 28.8 and 46.7% and induced apoptosis by 30 and 54%, respectively. For the first time, we reported that rosmarinic acid has the ability to trigger caspase-3 induced apoptosis in human glioma cells. As a result of siRNA transfection, the Hsp27 gene was silenced by ~?50% but did not cause a statistically significant change in caspase-3 activation. It was also observed that apoptosis was induced at a higher level as a result of Hsp27 siRNA and subsequent quercetin or RA treatment. siRNA transfection and 215 μM RA treatment suppressed Hsp27 expression level by 90.5% and increased caspase-3 activity by 58%. Herein, we demonstrated that RA administered with siRNA seems to be a potent combination for glioblastoma therapy.     

The NAD(P)H:Quinone Oxidoreductase 1 induces cell cycle progression and proliferation of melanoma cells

The oxidoreductase NQO1 plays a prominent role in maintaining the cellular homeostasis. NQO1 is mainly a cytosolic enzyme which catalyzes the metabolism of quinones and is present in almost all tissue types providing protection against different stresses including xenobiotics, oxidants, UV light, and ionizing radiation. This enzyme is overexpressed in many cancerous tissues and its function in carcinogenesis remains unclear. Due to the relative lack of information on the role of NQO1 in melanoma pathogenesis, we attempted to determine the expression and basic function of NQO1 in melanoma cell proliferation. We found that NQO1 is overexpressed in most melanoma cell lines with respect to melanocytes. Furthermore, the expression of this oxidoreductase significantly induces cell cycle progression by upregulating the expression of cyclins A2, B1 and D1, leading to the proliferation of melanoma cells. Our results also indicate that NQO1 is an upstream regulator of NF-κB p50, a factor linked to melanoma progression and poor patient prognosis. Interestingly, we found that NQO1 stabilizes the transactivator BCL3, which in turn upregulates NF-κB p50. More importantly, our results also indicate that NF-κB p50 correlates with the expression of NQO1 and mediates its role in the proliferation of melanoma cells.     

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.