Youhei Saito

Hsp105 but not Hsp70 family proteins suppress the aggregation of heat‐denatured protein in the presence of ADP

Hsp105α and Hsp105β are mammalian members of the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Here, we show that Hsp105α and Hsp105β bind non‐native protein through the β‐sheet domain and suppress the aggregation of heat‐denatured protein in the presence of ADP rather than ATP. In contrast, Hsc70/Hsp40 suppressed the aggregation of heat‐denatured protein in the presence of ATP rather than ADP. Furthermore, the overexpression of Hsp105α but not Hsp70 in COS‐7 cells rescued the inactivation of luciferase caused by ATP depletion. Thus, Hsp105/110 family proteins are suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly.

Decreased expression of endoplasmic reticulum chaperone GRP78 in liver of diabetic mice.

To identify molecular targets associated with the development of diabetes, we analyzed the hepatic proteome of obese diabetic db/db mice using electrophoresis on a high-resolution two-dimensional gel combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. By comparison between non-diabetic db/+ and diabetic db/db mice, six proteins and one protein were significantly decreased and increased in the diabetic mice, respectively. Among these proteins, two of the decreased proteins are involved in endoplasmic reticulum (ER) stress-related unfolded protein response, GRP78 and protein disulfide isomerase A3, and it was revealed that the decreased GRP78 expression in the liver of diabetic db/db mice is due to the reduction of GRP78 protein synthesis rather than RNA transcription. In addition, we found that the treatment of human hepatocyte HepG2 cells with oleic acid decreased the expression of GRP78, and attenuated the activation of AKT by insulin stimulation. These results suggest that decreased GRP78 expression may induce resistance to insulin by inhibiting the AKT activation, and plays an important role in the development of type 2 diabetes.

Hsp105β upregulates hsp70 gene expression through signal transducer and activator of transcription-3

Hsp105α and Hsp105β are mammalian members of the Hsp105/110 family, a divergent subgroup of the Hsp70 family. Hsp105α is expressed constitutively and induced by various forms of stress, whereas Hsp105β is an alternatively spliced form of Hsp105α that is expressed specifically during mild heat shock. In a report, it was shown that Hsp105α and Hsp105β localize to the cytoplasm and of nucleus of cells, respectively, and that Hsp105β, but not Hsp105α, induces the expression of Hsp70 in mammalian cells. Here, we examined the mechanism by which Hsp105β induces the expression of Hsp70. Using a series of deletion mutants of Hsp105β, it was revealed that the region between amino acids 642 and 662 of Hsp105β is necessary for the activation of the hsp70 promoter by Hsp105β. Furthermore, it was shown that signal transducer and activator of transcription (STAT)‐3 bound to the sequence of the hsp70 promoter between −206 and −187 bp, and that mutations of this sequence abrogated the activation of the hsp70 promoter by Hsp105β. In addition, overexpression of Hsp105β stimulated the phosphorylation of STAT3 at Tyr705 and its translocation to the nucleus. Downregulation of STAT3 expression resulted in reduction of the activation of the hsp70 promoter by Hsp105β. Furthermore, downregulation of Hsp105β reduced the expression of Hsp70 in heat‐shocked cells. On the basis of these findings, it is suggested that Hsp105β induces Hsp70 expression markedly through the STAT3 pathway in heat‐shocked cells. This may represent the mechanism that connects the heat shock protein and STAT families for cell defense against deleterious stress.

Nuclear Localization Mechanism of Hsp105β and its Possible Function in Mammalian Cells

Hsp105alpha and Hsp105beta are mammalian stress proteins of the Hsp105/110 family. We have shown that Hsp105beta localizes to the nucleus, whereas Hsp105alpha localizes to the cytoplasm of mammalian cells. Hsp105alpha localizes in the cytoplasm, as the nuclear export signal (NES) activity rather than nuclear localization signal (NLS) activity dominates in Hsp105alpha, due to suppression of the NLS activity. In this study, we determined the mechanisms behind the nuclear localization of Hsp105beta, and revealed that the NES was suppressed by the N-terminal (amino acids 3-10) or C-terminal (amino acids 699-756) region of Hsp105beta, and the NLS activity rather than NES activity seemed to dominate in Hsp105beta. Furthermore, as Hsp105beta which localizes in the nucleus, functioned as an inducer of Hsp70 in mammalian cells, Hsp105 family proteins may play an important role in the protection of cells against deleterious stressor together with Hsp70.

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells

Hsp105α and Hsp105β are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has opposite effects on stress‐induced apoptosis depending on the cell type. However, it is not fully understood how Hsp105 regulates stress‐induced apoptosis. In this study, we examined how Hsp105α and Hsp105β regulate H2O2‐induced apoptosis by using HeLa cells in which expression of Hsp105α or Hsp105β was regulated using doxycycline. Overexpression of Hsp105α and Hsp105β suppressed the activation of caspase‐3 and caspase‐9 by preventing the release of cytochrome c from mitochondria in H2O2‐treated cells. Furthermore, both c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (p38 MAPK) were activated by treatment with H2O2, and the activation of both kinases was suppressed by overexpression of Hsp105α and Hsp105β. However, H2O2‐induced apoptosis was suppressed by treatment with a potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor, SP600125. These findings suggest that Hsp105α and Hsp105β suppress H2O2‐induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis.

Identification of α-tubulin as an hsp105α-binding protein by the yeast two-hybrid system

Hsp105α is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105α prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105α, we searched for Hsp105α-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105α using the yeast two-hybrid system and obtained α-tubulin as an Hsp105α-binding protein. Hsp105α bound directly to α-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-α-tubulin antibodies indicated that Hsp105α was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105α-overexpressing COS-7 cells. These findings suggested that Hsp105α associates with α-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105alpha is expressed constitutively in the cytoplasm of cells, while Hsp105beta, an alternatively spliced form of Hsp105alpha, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105beta but also Hsp105alpha accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105alpha and Hsp105beta with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105alpha and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105alpha and Hsp105beta on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105alpha and Hsp105beta suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.

Arctigenin from Fructus Arctii is a novel suppressor of heat shock response in mammalian cells

Abstract Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoter-regulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy.

Genistein Induces Cytokinesis Failure Through RhoA Delocalization and Anaphase Chromosome Bridging

Genistein, an isoflavone abundantly present in soybeans, possesses anticancer properties and induces growth inhibition including cell cycle arrest and apoptosis. Although abnormal cell division, such as defects in chromosome segregation and spindle formation, and polyploidization have been described, the mechanisms underlying the induction of abnormal cell division are unknown. In this study, we examined the effect of genistein on cell division in cells that are synchronized in M phase, since genistein treatment delays mitotic entry in asynchronous cells. HeLa S3 cells were arrested at the G2 phase and subsequently released into the M phase in presence of genistein. Immunofluorescence staining showed that genistein treatment delays M phase progression. Time‐lapse analysis revealed that the delay occurs until anaphase onset. In addition, genistein treatment induces cleavage furrow regression, resulting in the generation of binucleated cells. Central spindle formation, which is essential for cytokinesis, is partially disrupted in genistein‐treated cells. Moreover, aberrant chromosome segregation, such as a chromosome bridge and lagging chromosome, occurs through progression of cytokinesis. RhoA, which plays a role in the assembly and constriction of an actomyosin contractile ring, is delocalized from the cortex of the ingressing cleavage furrow. These results suggest that genistein treatment induces binucleated cell formation through cleavage furrow regression, which is accompanied by chromosome bridge formation and RhoA delocalization. Our results provide the mechanism that underlies genistein‐induced polyploidization, which may be involved in genistein‐induced growth inhibition. J. Cell. Biochem. 115: 763–771, 2014.