Hsp60 Protects against Amyloid ß Oligomer Synaptic Toxicity via Modification of Toxic Oligomer Conformation.

Alzheimer's disease (AD) is the leading cause of dementia worldwide. While the etiology of AD remains uncertain, neurotoxic effects of amyloid beta oligomers (Aßo) on synaptic function, a well-established early event in AD, is an attractive area for the development of novel strategies to modify or cease the disease's progression. In this work, we tested the protective action of the mitochondrial chaperone Hsp60 against Aßo neurotoxicity, by determining the direct effect of Hsp60 in changing Aßo toxic conformations and thus reducing their dysfunctional synaptic binding and consequent suppression of long-term potentiation. Our data suggest that Hsp60 has a direct impact on Aßo, resulting in a reduction of cytotoxicity and rescue of Aßo-driven synaptic damage, thus proposing Hsp60 as an attractive therapeutic target candidate.

A rationally designed peptide IA-2-P2 against type 1 diabetes in streptozotocin-induced diabetic mice.

Recent studies have investigated the potential of type 1 diabetes mellitus-related autoantigens, such as heat shock protein 60, to induce immunological tolerance or to suppress the immune response. A functional 24-residue peptide derived from heat shock protein 60 (P277) has shown anti-type 1 diabetes mellitus potential in experimental animals and in clinical studies, but it also carries a potential atherogenic effect. In this study, we have modified P277 to retain an anti-type 1 diabetes mellitus effect and minimize the atherogenic potential by replacing the P277 B epitope with another diabetes-associated autoantigen, insulinoma antigen-2 (IA-2), to create the fusion peptide IA-2-P2. In streptozotocin-induced diabetic C57BL/6J mice, the IA-2-P2 peptide displayed similar anti-diabetic effects to the control P277 peptide. Also, the IA-2-P2 peptide did not show atherogenic activity in a rabbit model. Our findings indicate the potential of IA-2-P2 as a promising vaccine against type 1 diabetes mellitus.

Bacterial Heat Shock Protein GroEL (Hsp64) Exerts Immunoregulatory Effects on T Cells by Utilizing Apoptosis.

Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis.

Mycobacterial heat shock protein 65 (mbHSP65)-induced atherosclerosis: Preventive oral tolerization and definition of atheroprotective and atherogenic mbHSP65 peptides.

OBJECTIVE: The aim of this study was to identify atherogenic and atheroprotective peptides of bacterial HSP60 [taking mycobacterial HSP65 (mbHSP65) as a potent paradigmatic representative] that could be used as candidates for an orally applied tolerizing vaccine against atherosclerosis. METHODS: ApoE(-/-) mice were immunized with mbHSP65 protein or peptides, given mbHSP65 orally and then kept either on chow or high cholesterol diet. Atherosclerosis was assessed by en face and immunohistological analysis. Anti-HSP autoantibodies were detected by ELISA. The number and in vitro suppressive function of splenic and lymph node regulatory T cells (Tregs) were analyzed by flow cytometry. Specific T cell reactivity against mbHSP65 protein or peptides was assessed by proliferation assay. RESULTS: Decreased lesion size was accompanied by (a) increased splenic Treg numbers; (b) increased interleukin (IL)-10 mRNA levels in the aorta; (c) increased levels of anti-mbHSP65 and anti-mouse HSP60 antibodies pointing to pro-eukaryotic HSP60 humoral crossreaction, not curtailed by oral tolerization; (d) most importantly, we identified and functionally characterized novel atherogenic and atheroprotective mbHSP65 epitopes. CONCLUSION: Atheroprotective mbHSP65 peptides may be considered as potential candidates for the development of a tolerizing vaccine to prevent and treat atherosclerosis, while keeping protective immunity to non-atherogenic domains of mbHSP65 intact.

Deconstruction of stable cross-Beta fibrillar structures into toxic and nontoxic products using a mutated archaeal chaperonin.

Our group recently determined that a mutant archaeal chaperonin (Hsp 60) exhibited substantially enhanced protein folding activity at low temperatures and was able to deconstruct refractory protein aggregates. ATP dependent conversion of fibril structures into amorphous aggregates was observed in insulin amyloid preparations (Kurouski et al. Biochem. Biophys. Res. Commun. 2012). In the current study, mechanistic insights into insulin fibril deconstruction were obtained by examination of early stage complexes between Hsp60 and fibrils in the absence of ATP. Activity of the Hsp60 was significantly curtailed without ATP; however, some fibril deconstruction occurred, which is consistent with some models of the folding cycle that predict initial removal of unproductive protein folds. Chaperonin molecules adsorbed on the fibril surface and formed chaperonin clusters with no ATP present. We propose that there are specific locations on the fibril surface where chaperonin can unravel the fibril to release short fragments. Spontaneous coagulation of these fibril fragments resulted in the formation of amorphous aggregates without the release of insulin into solution. The addition of ATP significantly increased the toxicity of the insulin fibril-chaperonin reaction products toward mammalian cells.

Chlamydia heat shock protein 60 decreases expression of endothelial nitric oxide synthase in human and porcine coronary artery endothelial cells.

AIMS: Clinically, Chlamydia pneumoniae infection and its heat shock protein 60 (cHSP60) may contribute to atherogenesis; however, its underlying mechanisms are largely unknown. The objective of this study was to determine whether cHSP60 could cause endothelial dysfunction in human coronary artery endothelial cells (HCAECs) and porcine coronary arteries. METHODS AND RESULTS: When HCAECs were treated with recombinant cHSP60, endothelial nitric oxide synthase (eNOS) mRNA and protein levels, enzyme activities, cellular NO levels, mRNA stability, and promoter activities were significantly decreased. Superoxide anion production was significantly increased due to the inhibition of mitochondrial membrane potential and catalase and superoxide dismutase (SOD) activities as well as activation of NADPH oxidase. Antioxidant seleno-l-methionine (SeMet) or SOD mimetic MnTBAP effectively blocked cHSP60-induced eNOS downregulation. In addition, cHSP60 activated mitogen-activated protein kinases (MAPKs) including p38, c-Jun-N-terminal kinase/stress-activated protein kinase, and extracellular signal-regulated kinases. Specific chemical inhibitors or their dominant-negative mutant forms of these MAPKs effectively blocked cHSP60-induced eNOS downregulation. cHSP60-induced eNOS downregulation and oxidative stress were also demonstrated in porcine coronary artery rings in vitro. Functionally, endothelium-dependent vasorelaxation was significantly reduced in cHSP60-treated vessels. CONCLUSION: cHSP60 directly induces eNOS downregulation through oxidative stress and MAPK activation in both HCAECs and porcine coronary arteries, thereby causing endothelial dysfunction.

The GroEL-like protein from Campylobacter rectus: immunological characterization and interleukin-6 and -8 induction in human gingival fibroblast.

The native GroEL-like protein was purified from Campylobacter rectus, a putative periodontal pathogen, by affinity chromatography on ATP-agarose followed by high performance liquid chromatography on Superose 6. The purified 64-kDa protein (denatured form of GroEL-like protein) was immunoreactive by SDS-PAGE and Western immunoblotting with the monoclonal antibody directed against heat shock protein 60 of human origin. The native GroEL-like protein stimulated both interleukin-6 (IL-6) and IL-8 secretion by a confluent monolayer of human gingival fibroblast in their culture supernatant. During the 22-h incubation, the amounts of IL-6 and IL-8 were increased by 5.4- and 3.5-fold, respectively. These data suggested that the GroEL-like protein might be considered to be a virulence factor of C. rectus in periodontal disease.

Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans.

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.