Protective effects of small heat shock proteins in Daphnia magna against heavy metal exposure.

Daphnia magna is one of the most commonly used model organisms to assess toxicity of heavy metal and other xenobiotics. However, the lack of knowledge about important stress-resistant molecules limits our understanding of the alteration of phenotypic and physiological traits of D. magna upon stress exposures. In this study, we focused on a chaperone family of small heat shock protein (sHSP) that has been found in archaea, bacteria and eukaryotes and plays an important role in stress tolerance. A total of eleven sHSP genes (termed DmsHSP1 - DmsHSP11) were identified from the D. magna genome, whose expression profiles during exposure to heavy metal (Cd2+, Cu2+ and Zn2+) and a few other potential pollutants were evaluated via qRT-PCR and RNA-Seq analysis. The results highlighted the predominant role of DmsHSP1 with the highest basal expression level in adults and robust upregulation upon exposure to heavy metals (Cu2+ > Cd2+ > Zn2+). In vivo, recombinant protein rDmsHSP1-21 and rDmsHSP11-12.8 could not only prevent model substrates agglutination induced by heavy metals or reducer dithiotreitol (DTT), but also protect tissue proteins and enzymes from denaturation and inactivation caused by heavy metals or high temperature. Ectopically expression of DmsHSP1-21 or DmsHSP11-12.8 in E. coli conferred host enhanced resistance against various abiotic stresses including Cd2+, Cu2+ and phenazine methosulfate (PMS). Knockdown of DmsHSP1-21 by RNAi, but not for DmsHSP11-12.8, significantly increased the vulnerability of D. magna to heavy metal exposure. Our work provides systematic information on the evolution and function of sHSPs in D. magna and leads to important insights into the mechanisms by which D. magna survive in adverse environments.

Extracellular vesicle-associated small heat shock proteins as therapeutic agents in neurodegenerative diseases and beyond.

Increasing evidence points towards using extracellular vesicles (EVs) as a therapeutic strategy in neurodegenerative diseases such as multiple sclerosis, Parkinson's, and Alzheimer's disease. EVs are nanosized carriers that play an essential role in intercellular communication and cellular homeostasis by transporting an active molecular cargo, including a large variety of proteins. Recent publications demonstrate that small heat shock proteins (HSPBs) exhibit a beneficial role in neurodegenerative diseases. Moreover, it is defined that HSPBs target the autophagy and the apoptosis pathway, playing a prominent role in chaperone activity and cell survival. This review elaborates on the therapeutic potential of EVs and HSPBs, in particular HSPB1 and HSPB8, in neurodegenerative diseases. We conclude that EVs and HSPBs positively influence neuroinflammation, central nervous system (CNS) repair, and protein aggregation in CNS disorders. Moreover, we propose the use of HSPB-loaded EVs as advanced nanocarriers for the future development of neurodegenerative disease therapies.

Tumor microenvironment targeting with dual stimuli-responsive nanoparticles based on small heat shock proteins for antitumor drug delivery.

Tumour microenvironment (TME)-targeting nanoparticles (NPs) were developed based on Methanococcus jannaschii small heat shock proteins (Mj-sHSPs). Transactivator of transcription (TAT) were modified on the surface of Mj-sHSPs (T-HSPs) to enhance their cellular internalization ability (CIA), and a pH/enzyme dual sensitive PEG/N-(2-aminoethyl)piperidine-hyaluronic acid (PAHA) coat was combined with T-HSPs (PT-HSPs). PT-HSP NPs exhibited multi-layered morphologies and good stability against plasma protein adsorption. The release of paclitaxel (PTX) from PT-HSP NPs was negligible at physiological pH. Under conditions similar to the TME (acidic pH and overexpressed hyaluronidase (HAase)), the PAHA coat deshielded from PT-HSP NPs because of two factors: charge reversal and HAase degradation. Once the PAHA coat was shed, the size of the NPs decreased; its surface charge became positive; and remarkable drug release was triggered. Cellular experiments indicated that the CIA of PT-HSPs was shielded in the microenvironment of normal cells and recovered in that of tumour cells. In vivo imaging exhibited that the PT-HSP NPs had an impressive tumour targeting ability compared with the uncoated controls. The antitumor efficacy in vivo demonstrated that tumour-bearing mice treated with PTX-loaded PT-HSP NPs achieved better anti-tumour effects and safety than the Taxol formulation. In summary, this study provided Mj-sHSP NPs with coats that could be shed in response to the particular pH and enzymes in the TME, which improved the efficacy of tumour therapy. STATEMENT OF SIGNIFICANCE: This study reports on tumor microenvironment-targeting protein-based nanoparticles (PT-HSP NPs) for targeted tumor therapy. The NPs had a multilayered structure: a protein cage, a TAT cationic layer, and a dual-sensitive coat. PT-HSP NPs exhibited multilayered morphology, with good stability against plasma protein adsorption, and PTX release negligible at physiological pH. Under the tumor microenvironment (acidic pH and overexpressed HAase), PAHA coat deshielded from PT-HSP NPs due to two factors: the charge reversal induced by protonation of piperidines in PAHA and HAase degradation. The results of cellular uptake, cytotoxicity, in vivo imaging, and tumor inhibition experiments confirmed that PT-HSP NPs exhibited promising tumor targeting efficacy in vitro and in vivo.

Small heat shock protein AgsA: an effective stabilizer of enzyme activities.

A small heat shock protein, AgsA, possesses chaperone activity that can reduce the amount of heat-aggregated protein in vivo, and suppress the aggregation of chemical- and heat-denatured proteins in vitro. Therefore, we examined the ability of AgsA to stabilize the activity of several enzymes by using this chaperone activity. We observed that AgsA can stabilize the enzymatic activities of Renilla (Renilla reniformis) luciferase, firefly (Photinus pyralis) luciferase, and ß-galactosidase, and showed comparable or greater stabilization of these enzymes than bovine serum albumin (BSA), a well-known stabilizer of enzyme activities. In particular, AgsA revealed better stabilization of Renilla luciferase and ß-galactosidase than BSA under disulfide bond-reducing conditions with dithiothreitol. In addition, AgsA also increased the enzymatic performance of ß-galactosidase and various restriction enzymes to a comparable or greater extent than BSA. These data indicate that AgsA may be useful as a general stabilizer of enzyme activities.

Cell permeant peptide analogues of the small heat shock protein, HSP20, reduce TGF-beta1-induced CTGF expression in keloid fibroblasts.

A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

Heat-shock protein 25 ameliorates calcium oxalate crystal-mediated oxidative stress in renal epithelial cells.

OBJECTIVE: To investigate whether the antioxidant protection attributed to small heat-shock proteins (sHSPs) affects calcium oxalate stone formation, a pro-oxidant disease. MATERIALS AND METHODS: Canine distal tubular epithelial cells (Madin-Darby canine kidney, MDCK cells) were grown as confluent monolayers. Treatment regimens included control and HS-treated cells (37 degrees C and 42 degrees C for 1 h) with or without calcium oxalate monohydrate (COM) or free oxalate treatment (28 microg/cm2) 16 h later. In digitonin-permeabilized cells, O2- was measured by lucigenin-enhanced chemiluminescence over a 5-min period, to measure mitochondrial O2- production. Protein expression was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis Western blot analysis using specific antibodies. RESULTS: COM significantly increased O2- production in MDCK cells. HS treatment, which up-regulated HSP25 expression, significantly decreased this O2- production (P < 0.05) but had no effect in control cells. In COM-treated cells (20 h) there was a marked and significant down-regulation of both HSP 25, HSP 70 and heme oxygenase-1 expression compared to cells treated with HS alone (P < 0.05). Free oxalate had no effect on HSP 25 expression. CONCLUSIONS: The results suggest that the COM-induced increase in mitochondrial O2- production in MDCK cells is ameliorated by HSP 25 up-regulation via HS. Specific COM inhibition of HSP 25, HSP 70 and heme oxygenase-1 up-regulation suggests that COM-induced reactive oxygen species damage is unable to benefit from HSP-associated physiological resistance.

Small heat shock proteins differentially affect Abeta aggregation and toxicity.

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.

The possibility of novel antiplatelet peptides: the physiological effects of low molecular weight HSPs on platelets.

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported on a physiological role of HSP20, HSP27 and alphaB-crystallin on platelet function in vitro and ex vivo. HSP20 and alphaB-crystallin inhibited platelet aggregation using human platelets dose-dependently induced by thrombin or botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When HSP20 or alphaB-crystallin was injected intravenously as a bolus in hamsters, the development of thrombus after endothelial injury was prevented. Moreover, 9 amino-acid sequences isolated from HSP20 or alphaB-crystallin significantly reduced platelet aggregation induced by TRAP, but not a PAR-4 agonist. These findings strongly suggest that HSP20 or alphaB-crystallin can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation in vivo.

A small HSP, Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium.

The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytoplasm. In order to determine if Lo18 could interact with the phospholipids, we used model membranes made of lipids extracted from O. oeni cells. Using fluorescence anisotropy of diphenylhexatriene (DPH) and generalized polarization of Laurdan, we showed that purified Lo18 interacts with these liposomes, and increases the molecular order of the lipid bilayer in these membranes when the temperature reaches 33.8 degrees C. All these data suggest that Lo18 could be involved in an adaptive response allowing the maintenance of membrane integrity during stress conditions in O. oeni cells.