Role for NAD(P)H:quinone oxidoreductase 1 and manganese-dependent superoxide dismutase in 17-(allylamino)-17-demethoxygeldanamycin-induced heat shock protein 90 inhibition in pancreatic cancer cells

Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH?). The formation of 17AAGH? by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH? were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH? could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH?), reduction of 17AAG to 17AAGH? by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH?. In addition, oxidation of 17AAGH? could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH? was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH? with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH?, the active Hsp90 inhibitor derived from 17AAG.     

Enhancement of periosteal bone formation by basic fibroblast‐derived growth factor containing polycystic kidney disease and collagen‐binding domains from Clostridium histolyticum collagenase

Recombinant basic fibroblast growth factor (bFGF) is a potent mitogen for mesenchymal cells that accelerates bone union and repair when applied locally at defect sites. However, because bFGF diffuses rapidly from bone defect sites, repeated dosing is required for sustained therapeutic effect. We previously fused the collagen‐binding domain (CBD) and polycystic kidney disease (PKD) domain of Clostridium histolyticum class II collagenase (ColH) to bFGF and demonstrated that the fusion protein markedly enhances bone formation when loaded onto collagen materials used for grafting. However, systemic injection of a fusion protein consisting of parathyroid hormone (PTH) and a CBD was shown to accelerate bone formation in an osteoporosis model more rapidly than treatment with a PTH–PKD–CBD fusion protein. Here, we compared the biological properties of two collagen‐binding forms of bFGF, bFGF–CBD and bFGF–PKD–CBD. Both fusion proteins promoted the in vitro proliferation of periosteal mesenchymal cells, indicating that they had biological activity similar to that of native bFGF. In vivo periosteal bone formation assays in rat femurs showed that both bFGF–CBD and bFGF–PKD–CBD induced periosteal bone formation at higher rates than collagen sheet alone and bFGF. However, bFGF–PKD–CBD markedly enhanced bone formation and had higher collagen‐binding ability than bFGF–CBD in in vitro protein release assays. Taken together, these results suggest that the PKD domain increases the retention of bFGF at graft sites by enhancing collagen‐binding affinity. Therefore, bFGF–PKD–CBD–collagen composite appears to be a promising material for bone repair in the clinical setting. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of cuticular chitin‐binding proteins of Leptinotarsa decemlineata (Say) and post‐ecdysial transcript levels at different developmental stages

Seven cuticle chitin‐binding proteins (Ld‐CP1v1 to 7) were deduced from antenna cDNAs of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), based on their consensus sequences. The mature proteins consisted of 87–188 residues. Ld‐CP1v1 formed a distinct orthologous protein cluster (OP1) along with four proteins from other insect species in a neighbor‐joining phylogenetic tree. These proteins also contained a proline glutamine‐rich (PQ‐rich) region and a highly conserved C‐terminal motif (Phr). Their consensus region lacked the defined aromatic triad. Ld‐CP2 to 6 clustered with those bearing RR‐1 consensus and Ld‐CP7 with RR‐2 consensus. Ld‐CP1v1 to 4 were expressed at the post‐ecdysial period in all the developmental stages whereas Ld‐CP5 to 7 were expressed mainly in adults.     

Use of affinity‐directed liquid chromatography‐mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

Summary. Background: Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods: Reactive epitopes to an immobilized IgG fraction prepared from a high‐titer, FVIII inhibitor plasma were determined after immuno‐precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography‐mass spectrometry (LC‐MS). Results: Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC‐MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues. Conclusions: IP coupled with LC‐MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein–protein and protein–membrane interactions.     

Bombyx mori cyclin‐dependent kinase inhibitor is involved in regulation of the silkworm cell cycle

Cyclin‐dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin‐dependent kinase (CDK)‐cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654‐bp‐long BmCKI‐L (the longer splice variant) encoding a protein with 217 amino acids and a 579‐bp‐long BmCKI‐S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI‐L and BmCKI‐S contain the Cip/Kip family conserved cyclin‐binding domain and the CDK‐binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181–210. Overexpression of BmCKI‐L or BmCKI‐S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI‐L or BmCKI‐S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI‐L and BmCKI‐S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI‐L overexpression (BmCKI‐L‐OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI‐L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm.     

Evaluation of prostate-specific antigen as a novel biomarker of Hsp90 inhibition

ObjectivesHsp90 inhibition has already been evaluated by assessing the expression of proteins, like the androgen receptor (AR) and the IGFBP-2. We hypothesized that AR-regulated proteins, such as prostate-specific antigen (PSA), may serve as biomarkers for Hsp90 inhibition.Design and methodsWe utilized the androgen-stimulated BT-474 breast cancer cells to trigger PSA secretion, which was quantified by ELISA. PSA concentration was used to evaluate the potency of an experimental compound (NVP-AUY922), in comparison to the commercially available 17-allylamine-17-demethoxygeldanamycin (17-AAG) Hsp90 inhibitor.ResultsPSA concentration was reduced in a dose-dependent manner (2-fold more than IGFBP-2) and was accompanied by AR decrease. Utilizing PSA expression as a marker for Hsp90 inhibition, we concluded that the novel NVP-AUY922 inhibitor was about 8-fold more potent than 17-AAG.ConclusionsThis study showed that PSA may serve as a sensitive biomarker of Hsp90 inhibition and may aid in selecting new chemotherapeutics. Furthermore, the novel Hsp90 inhibitor was highly potent, suggesting that it may be an attractive agent for clinical trials.     

A MC motif in silkworm Argonaute 1 is indispensible for translation repression

Small RNA‐mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA‐induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double‐stranded RNA (dsRNA)‐induced RNAi, whereas Ago3 and Siwi are involved in the Piwi‐interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)‐mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA‐decapping enzyme 2 (Dcp2) protein in the Drosophila processing‐bodies (P‐bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P‐bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.     

Plasma antibody titres to heat shock proteins‐60, ‐65 and‐70: Their relationship to coronary risk factors in dyslipidaemic patients and healthy individuals

Objective To investigate the factors that may affect antibody titres to heat shock proteins (Hsp)‐60, ‐65 and ‐70, and serum C‐reactive protein (CRP) concentrations in patients with dyslipidaemia and other features of the metabolic syndrome as defined by ATPIII criteria. Material and methods. The study comprised 237 dyslipidaemia patients and 135 healthy individuals recruited from amongst university and hospital employees. Results. Compared to the healthy individuals, the dyslipidaemic patients had higher antibody titres to Hsp‐60 (p<0.01), Hsp‐65 (p<0.001) and Hsp‐70 (p<0.05), and higher serum CRP concentrations (p<0.001). The best‐fitting multifactorial models revealed that known coronary risk factors explained little of the variation in Hsp antibody titres: 3 % for Hsp‐60, 1 % for Hsp‐65 and 4 % for Hsp‐70 amongst the dyslipidaemic subjects. The corresponding values for the subgroup with the metabolic syndrome were 8 %, 3 % and 1 %, respectively. In contrast, the best‐fitting model explained 13.5 % of the variation in serum CRP concentrations among the dyslipidaemic patients, obesity being a major determinant; and 14 % in the subgroup with metabolic syndrome. Conclusions. The higher antibody titres to Hsp‐60, ‐65, and ‐70 in the dyslipidaemic patients may be related to a heightened state of immunoactivation associated with atherosclerosis in this group. Our data indicate that antibody titres to these Hsps are not associated with the classical coronary risk factors, although serum high sensitivity (hs)CRP concentrations were significantly related to obesity.     

Bioeffects of Low‐Intensity Ultrasound In Vitro

Objective. The purpose of this study was to evaluate the potential molecular mechanism of low‐intensity ultrasound‐induced apoptosis by analyzing protein profile alteration in response to ultrasound exposure. Methods. Human hepatocarcinoma SMMC‐7721 cells were used in this study. Cell viability was measured by a trypan blue dye exclusion test. Morphologic changes were examined by light microscopy. Apoptosis was assessed by phosphatidylserine externalization and DNA fragmentation. The pattern of the mitochondrial membrane potential decrease was determined by flow cytometry. Protein profile alteration was analyzed by comparative proteomics based on 2‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Results. Low‐intensity ultrasound (3.0 W/cm², 1 minute, cells incubated for 6 hours after ultrasound exposure) induced early apoptosis (mean ± SD, 26.5% ± 6.2%) significantly (P < .05) with minimal lysis in human hepatocarcinoma cells in vitro. On a molecular level, several proteins, eg, cellular tumor antigen protein 53, BH3‐interacting domain death agonist, apoptosis regulator Bcl‐2, and heme oxygenase 1 were identified as responding to ultrasound irradiation, suggesting that mitochondrial dysfunction and oxidative stresses were involved in ultrasound‐induced apoptosis. It was also assumed that mitofilin‐regulated crista remodeling may be a potential channel of mitochondrial membrane permeabilization pore formation involved in low‐intensity ultrasound‐induced apoptosis. Conclusions. This study suggests that 2 potential molecular signaling pathways are involved in ultrasound‐induced apoptosis. It is a first step toward low‐intensity ultrasound‐induced apoptotic cancer therapy via understanding its relevant molecular signaling and key proteins.     

Polyamine‐promoted autoactivation of plasma hyaluronan‐binding protein

See also Kanse SM, Etscheid M. Factor VII activating protease (FSAP): caught in the cross‐fire between polycations and polyanions. This issue, pp 556–8. Summary. Background: Plasma hyaluronan‐binding protein (PHBP), a protease implicated in extracellular proteolysis, consists of multiple domains: an N‐terminal region (NTR), three epidermal growth factor (EGF)‐like domains, a kringle domain, and a protease domain. PHBP circulates as a single‐chain proenzyme (pro‐PHBP), which is converted to an active, two‐chain form through autoproteolysis. Objective: To understand the mechanism of autoactivation. Here, we report that polyamine induces the formation of pro‐PHBP autoactivation complex, in which an intermolecular interaction between NTR and the third EGF‐like domain (E3) plays a role. Methods: Using a series of pro‐PHBP mutants that partially lack functional domains, polyamine‐induced pro‐PHBP autoactivation was investigated in terms of enzyme activity, protein interaction, and inhibition by carminic acid, an anthraquinone compound identified in this study. Results: Polyamine enhanced intermolecular binding of pro‐PHBP, but not of mutant pro‐PHBP that partially lacked NTR (ΔN). Carminic acid inhibited intermolecular pro‐PHBP binding and specifically abolished polyamine‐induced autoactivation. NTR bound to pro‐PHBP and ΔN, but its binding was minimal to a mutant that lacked E3. The NTR‐ΔN binding was inhibited by a combination of polyamine and carminic acid, but each compound alone was ineffective. Conclusions: We infer from the data that (i) polyamine modulates intramolecular NTR‐E3 interaction to allow intermolecular binding between NTR and E3 in another pro‐PHBP molecule to form an autoactivation complex, and (ii) carminic acid inhibits polyamine‐modulated intermolecular NTR‐E3 binding. Polyamine concentrations are higher in cells and tissues with inflammation and malignancy. Polyamine leakage from legions through cell death or tissue injury may account for physiologically relevant pro‐PHBP activation.     

A Drosophila heat shock response represents an exception rather than a rule amongst Diptera species

Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short‐lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold‐water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat‐resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.     

Insights into the molecular inactivation mechanism of human activated thrombin‐activatable fibrinolysis inhibitor

See also Gils A. Hot spots in TAFIa. This issue, pp 1054–5. Summary. Background: Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro‐domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half‐life of minutes.Objectives: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa. Results: We found that protein inhibitors significantly reduced the instability and insolubility of TAFIa. In addition, we solved the 2.5‐Å resolution crystal structure of human TAFIa in complex with a potent protein inhibitor, tick‐derived carboxypeptidase inhibitor, which gives rise to a stable and soluble TAFIa species. The structure revealed a significant reduction in the flexibility of dynamic segments when compared with the structures of bovine and human TAFI. We also identified two latent hotspots, loop Lβ2β3 and segment α5–Lα5β7–β7, where conformational destabilization may begin. These hotspots are also present in TAFI, but the pro‐domain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro‐domain is removed, the free TAFIa moiety becomes unstable, its activity is suppressed, and the molecule becomes insoluble. Conclusions: The present study corroborates the function of protein inhibitors in stabilizing human TAFIa and it provides a rigid and high‐resolution mold for the design of small molecule inhibitors of this enzyme, thus paving the way for novel therapy for thrombotic disorders.