Fermented food Tempeh induces interleukin 12 and enhances macrophage phagocytosis.

It is known that lactic acid bacteria induce the IL-12. The IL-12 activates NK cells and promotes the production of IFN-γ. The IFN-γ activates macrophages resulting in enhanced phagocytosis and bactericidal activity. We have been investigating fermented foods that activate the immune function. In this study, we investigated the IL-12 inducibility of fermented foods using the specific antibody. Fermented soybean foods such as Tempeh and Natto are attracting attention in terms of nutrition, functionality, and food problems. In this study, Tempeh induced 1,080 µg/ml of IL-12, and IFN-γ associated with the induction of IL-12 was also induced at 682 µg/ml. This was more than twice the induced intensity of PBS. On the contrary, Natto hardly induced IL-12 and IFN-γ. Tempeh also accelerated phagocytosis of the macrophage THP-1 cells. In this study, it was found that the fermented soybean-derived food, Tempeh, has a function of activating the immune function. This is the first report that Tempeh activates innate immunity. PRACTICAL APPLICATIONS: Tempeh, a fermented soybean food induced the IL-12 and IFN-γ production and the increase of macrophage phagocytosis in this study suggested a new function to enhance immunity. Tempeh is also expected to be effective in preventing lifestyle diseases. Fermented soybean products of Tempeh was considered to be a very useful health food for the problems of modern society such as maintaining health by eating, improving immunity, and ingesting vegetable protein due to diversifying food.

Biochemical properties of human full-length aryl hydrocarbon receptor (AhR).

The aryl hydrocarbon receptor (AhR) is a very unstable protein. AhR binds to the molecular chaperone complex (HSP90-p23-XAP2) to maintain a stable structure in the cytoplasm. After binding to ligands, such as dioxin, AhR translocates from the cytoplasm to the nucleus with a molecular chaperone complex. The protein forms a heterodimer with Arnt after nuclear transfer, functions as a transcription factor by binding to a xenobiotic responsive element (XRE), and induces the cytochrome P450 1A1 (CYP1A1). Because of the unstable protein, expression of the full-length AhR in the E. coli expression system is very difficult. Many studies investigated AhR using AhR domains in vitro. We expressed and purified the human full-length AhR in E. coli expression system. Furthermore, specific antibodies were prepared. Purified full-length AhR could bind to ligand. In the presence of ligand, α-helix and random coil of AhR increased and ß-sheet decreased on CD spectrum. Full-length AhR could bind to HSP90, XAP2 and p23 in the presence or absence of ligand. We now show the biochemical properties of full-length AhR.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

The regulation mechanisms of AhR by molecular chaperone complex.

The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

HSP60 possesses a GTPase activity and mediates protein folding with HSP10.

The mammalian molecular chaperone, HSP60, plays an essential role in protein homeostasis through mediating protein folding and assembly. The structure and ATP-dependent function of HSP60 has been well established in recent studies. After ATP, GTP is the major cellular nucleotide. In this paper, we have investigated the role of GTP in the activity of HSP60. It was found that HSP60 has different properties with respect to allostery, complex formation and protein folding activity depending on the nucleoside triphosphate present. The presence of GTP slightly affected the ATPase activity of HSP60 during protein folding. These results provide clues as to the functional mechanism of the HSP60-HSP10 complex.

High dose of antibiotic colistin induces oligomerization of molecular chaperone HSP90.

Colistin is an antimicrobial cationic peptide that belongs to the polymyxin family. Colistin was clinically used for the treatment of gram-negative infections but fell out of favour because of its significant side effects including neurotoxicity and nephrotoxicity. More recently, colistin has been regarded as one of the important options for nosocomial infections caused by multidrug resistant bacteria. Mechanisms of both the side effect onset of the drug and the side effect reduction are yet to be elucidated. In this study, we identified the specific binding protein of colistin using an affinity column chromatography. Colistin binds to the molecular chaperone HSP90. Although colistin slightly suppressed the chaperone activity of HSP90, there are no effects on the ATPase activity for a low concentration of colistin. Interestingly, colistin-induced aggregation of HSP90 via the N-domain. As for the cell viability of the SHSY5Y cell, the cell viability decreased to approximately 80% by the colistin 300 µM. However, the cell viability recovered to approximately 100% by adding ATP dosage. The same result was obtained by dot blot assay using anti-HSP90 antibody. Our results may help to understand the side effect mechanism of colistin.

Functional structure and physiological functions of mammalian wild-type HSP60.

The Chaperonins comprise a family of molecular chaperones having a double-ring structure and similar sequence homology. These proteins play an essential role in biological reactions that mediate the folding of newly synthesized polypeptides and partially denatured proteins. In the prokaryotic group I chaperonins, structural and reaction cycle analyses of GroEL and its co-chaperone GroES have been performed in detail. While in eukaryotes, there have been limited reports analyzing the group I chaperonin HSP60 and its co-chaperone HSP10. In the present study, we purified the wild type HSP60 from porcine liver and investigated the interaction between HSP60 and HSP10, including conformation and physiological relationships. Based on the results of transmission electron microscopy, native PAGE, and gel filtration column chromatography, the wild type HSP60 displayed a heptameric single-ring structure in the absence of ATP. In contrast, HSP60 formed mainly a "football-type" complex with HSP10 in the presence of ATP and mediated the refolding of denatured substrate protein. The functional conformation cycle of the purified mammalian HSP60 is distinct from the cycle of the prokaryotic GroEL/GroES chaperonin.

Geranylgeranylacetone selectively binds to the HSP70 of Helicobacter pylori and alters its coccoid morphology.

Geranylgeranylacetone (GGA) is used to treat patients suffering from peptic ulcers and gastritis. We examined the effect of GGA on Helicobacter pylori, which is a causative factor of gastrointestinal diseases. Previously, we have reported that GGA binds specifically to the molecular chaperone HSP70. In this paper, we report that GGA bounds to H. pylori HSP70 (product of the DnaK gene) with 26-times higher affinity than to human HSP70, and induced large conformational changes as observed from surface plasmon resonance and circular dichroism. Binding of GGA suppressed the activity of the H. pylori chaperone. GGA also altered several characteristics of H. pylori cells. GGA-treated cells elicited enhanced interleukin-8 production by gastric cancer cell lines and potentiated susceptibility to complement as compared to untreated cells. GGA also caused morphological alterations in H. pylori as reflected in fewer coccoid-like cells, suggesting that GGA converts H. pylori to an actively dividing, spiral state (vegetative form) from a non-growing, coccoid state. This morphological conversion by GGA resulted in accelerated growth of H. pylori. These results suggest a model in which GGA sensitizes H. pylori to antibiotic treatment by converting the cells to an actively growing state.

The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90.

The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or ß-naphthoflavone (ß-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to ß-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with ß-NF.

Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70.