Survey of malignant pleural mesothelioma treatment in Japan: Patterns of practice and clinical outcomes in tomotherapy facilities.

We conducted a nationwide survey of tomotherapy for malignant pleural mesothelioma (MPM) in Japan. Fifty-six facilities were surveyed and data on 31 patients treated curatively between 2008 and 2017 were collected from 14 facilities. Twenty patients received hemithorax irradiation after extrapleural pneumonectomy (EPP) (first group). Five patients received irradiation without EPP (second group), while six received salvage radiotherapy for local recurrence (salvage group). Among the seven patients not undergoing EPP, five (four in the second group and one in the salvage group) were treated with lung sparing pleural irradiation (LSPI) and two with irradiation to visible tumors. Two-year overall survival (OS) rates in the first and second groups were 33% and 60%, respectively (median, 13 vs 30 months, P = 0.82). In the first and second groups, 2-year local control (LC) rates were 53 and 67%, respectively (P = 0.54) and 2-year progression-free survival (PFS) rates were 16% and 60%, respectively (P = 0.07). Distant metastases occurred in 15 patients in the first group and three in the second group. In the salvage group, the median OS was 18 months. Recurrence was observed in the irradiated volume in four patients. The contralateral lung dose was higher in LSPI than in hemithorax irradiation plans (mean, 11.0 ± 2.2 vs 6.1 ± 3.1 Gy, P = 0.002). Grade 3 or 5 lung toxicity was observed in two patients receiving EPP and hemithorax irradiation, but not in those undergoing LSPI. In conclusion, outcomes of EPP and hemithorax irradiation were not satisfactory, whereas LSPI appeared promising and encouraging.

Structural and thermodynamical insights into the binding and inhibition of FIH-1 by the N-terminal disordered region of Mint3.

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted thermodynamic and structural studies in solution to clarify the structural and thermodynamical changes of Mint3 binding to FIH-1. First, using a combination of circular dichroism, nuclear magnetic resonance, and hydrogen/deuterium exchange-mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, we revealed a large enthalpy and entropy change in the interaction of Mint3 using isothermal titration calorimetry (ITC). The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified amino acids 78 to 88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part of Mint3 to the interaction with FIH-1, where the core sites contribute to the affinity (ΔG), while other sites only affect enthalpy (ΔH), by forming noncovalent bonds. This insight can serve as a foothold for further investigation of intrinsically disordered regions (IDRs) and drug development for cancer therapy.

Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel ß-Sheet Structure Formed by Amyloid ß-Protein on GM1 Clusters.

The abnormal aggregation of amyloid ß-protein (Aß) is considered central in the pathogenesis of Alzheimer's disease. We focused on membrane-mediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aß-(1-40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel ß-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single ß-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aß is included in the ß-sheet. Chemical cross-linking experiments using Cys-substituted Aßs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel ß-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

Use of a Compact Tripodal Tris(bipyridine) Ligand to Stabilize a Single-Metal-Centered Chirality: Stereoselective Coordination of Iron(II) and Ruthenium(II) on a Semirigid Hexapeptide Macrocycle.

Fe(II)-coordinating hexapeptides containing three 2,2'-bipyridine moieties as side chains were designed and synthesized. A cyclic hexapeptide having three [(2,2'-bipyridin)-5-yl]-d-alanine (d-Bpa5) residues, in which d-Bpa5 and Gly are alternately arranged with 3-fold rotational symmetry, coordinated with Fe(II) to form a 1:1 octahedral Fe(II)-peptide complex with a single facial-Λ configuration of the metal-centered chirality. NMR spectroscopy and molecular dynamics simulations revealed that the Fe(II)-peptide complex has an apparent C3-symmetric conformations on the NMR time scale, while the peptide backbone is subject to dynamic conformational exchange between three asymmetric ß/γ conformations and one C3-symmetric γ/γ/γ conformation. The semirigid cyclic hexapeptide preferentially arranged these conformations of the small octahedral Fe(II)-bipyridine complex, as well as the Ru(II) congener, to underpin the single configuration of the metal-centered chirality.

Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.

Fe(ii)-Complexation of tripodal hexapeptide ligands with three bidentate triazolylpyridines: induction of metal-centred chirality by peptide macrocyclization.

Fe(ii)-Coordinating peptides, having three bidentate 2-(1,2,3-triazol-4-yl)pyridine moieties at their side-chains, were designed based on the naturally occurring siderophore structures. The C3-symmetric macrocyclic peptide and an open-chain congener with the same sequence formed Fe(ii)-peptide complexes with opposite metal-centred chiralities.

The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates.

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.

Evaluation of the stability of an SR398/GroES chaperonin complex.

The stability of an SR398/GroES chaperonin complex was examined. As was expected, based on the finding of previous studies, the SR398/GroES complex was extremely stable in the presence of an excess amount of free adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-(ß,γ-imido)triphosphate (AMPPNP). However, the complex was not stable in the absence of nucleotides. These results indicate that ATPγS and AMPPNP repeatedly associated to and dissociated from the complex in a non-cooperative manner. This nucleotide exchange did not induce the dissociation of GroES and substrate from SR398, suggesting the importance of the cooperative dissociation of nucleotides from the cis-ring to release GroES and substrate proteins in the GroEL/GroES reaction cycle.

GM1 cluster mediates formation of toxic Aß fibrils by providing hydrophobic environments.

The conversion of soluble, nontoxic amyloid ß-proteins (Aß) to aggregated, toxic forms rich in ß-sheets is considered to be a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid-protein interactions play a crucial role in the aggregation of amyloidogenic proteins like Aß. Our group has previously reported that amyloid fibrils of Aß formed on membranes containing clusters of GM1 ganglioside (M-fibrils) exhibit greater cytotoxicity than fibrils formed in aqueous solution (W-fibrils) [ Okada ( 2008 ) J. Mol. Biol. 382 , 1066 - 1074 ]. W-fibrils are considered to consist of in-register parallel ß-sheets. However, the precise molecular structure of M-fibrils and force driving the formation of toxic fibrils remain unclear. In this study, we hypothesized that low-polarity environments provided by GM1 clusters drive the formation of toxic fibrils and compared the structure and cytotoxicity of W-fibrils, M-fibrils, and aggregates formed in a low-polarity solution mimicking membrane environments. First, we determined solvent conditions which mimic the polarity of raftlike membranes using Aß-(1-40) labeled with the 7-diethylaminocoumarin-3-carbonyl dye. The polarity of a mixture of 80% 1,4-dioxane and 20% water (v/v) was found to be close to that of raftlike membranes. Aß-(1-40) formed amyloid fibrils within several hours in 80% dioxane (D-fibrils) or in the presence of raftlike membranes, whereas a much longer incubation time was required for fibril formation in a conventional buffer. D-fibrils were morphologically similar to M-fibrils. Fourier-transform infrared spectroscopy suggested that M-fibrils and D-fibrils contained antiparallel ß-sheets. These fibrils had greater surface hydrophobicity and exhibited significant toxicity against human neuroblastoma SH-SY5Y cells, whereas W-fibrils with less surface hydrophobicity were not cytotoxic. We concluded that ganglioside clusters mediate the formation of toxic amyloid fibrils of Aß with an antiparallel ß-sheet structure by providing less polar environments.

Ganglioside-mediated aggregation of amyloid ß-proteins (Aß): comparison between Aß-(1-42) and Aß-(1-40).

Conversion of the soluble, non-toxic amyloid ß-protein (Aß) into an aggregated, toxic form rich in ß-sheets is considered a key step in the development of Alzheimer's disease. Accumulating evidence suggests that lipid rafts in membranes play a pivotal role in this process. We have proposed that Aß-(1-40) specifically bound to a ganglioside cluster forms cytotoxic fibrils via a conformational transition from an α-helix-rich structure to a ß-sheet-rich one. In the present study, we compared the interaction of Aß-(1-40) and Aß-(1-42) with both model and living cell membranes. Aß-(1-42) exhibited lipid specificity and affinity similar to Aß-(1-40), though its amyloidogenic activity was more than 10-fold that of Aß-(1-40). Antibody staining experiments, using the A11 antibody specific to Aß oligomers, demonstrated that oligomers were not detected during the aggregation process, and cell death was observed only after significant accumulation of the proteins, suggesting that the fibril-induced disruption of cell membranes leads to the cytotoxicity. Furthermore, we succeeded in visualizing fibrils formed on cell membranes using total internal reflection fluorescence microscopy. Aß-(1-40) formed long fibrils extruding to the aqueous phase, whereas Aß-(1-42) fibrils appeared to be laterally co-assembled and short.

Inhibitors of amyloid beta-protein aggregation mediated by GM1-containing raft-like membranes.

The aggregation (fibril formation) of amyloid beta-protein (Abeta) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Abeta aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Abeta aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Abeta aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Abeta to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Abeta in solution, thus at least partly preventing fibrils from forming. Coincubation of Abeta with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Abeta-induced toxicity in PC12 cells by inhibiting Abeta accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Abeta suggested that the two aggregation processes are not identical.

Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR.

Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.

Nuclear magnetic resonance characterization of the refolding intermediate of beta2-microglobulin trapped by non-native prolyl peptide bond.

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex type I, is also found as a major component of amyloid fibrils formed in dialysis-related amyloidosis. Denaturation of beta2-m is considered to initiate the formation of fibrils. To clarify the mechanism of fibril formation, it is important to characterize the intermediate conformational states at the atomic level. Here, we investigated the refolding of beta2-m from the acid-unfolded state by heteronuclear magnetic resonance and circular dichroism spectroscopies. At low temperature, beta2-m refolded slowly, accumulating a rate-limiting intermediate with non-native chemical shift dispersions for several residues, but with compactness and secondary structures similar to those of the native protein. beta2-m has a cis proline residue at Pro32, located on the turn connecting the betaB and betaC strands. The slow refolding phase disappeared upon mutation of Pro32 to Val, indicating that Pro32 is responsible for the accumulation of the intermediate. The distribution of the perturbed residues in the intermediate suggests that the non-native prolyl peptide bond of Pro32 affects large areas of the molecule. A cis proline residue is common to various immunoglobulin domains involved in amyloidosis, implying that a non-native prolyl peptide bond that might occur under physiological conditions is related to the amyloidogenicity of these immunoglobulin domains.

Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding.

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange.

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis.

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.

Conformational dynamics of beta(2)-microglobulin analyzed by reduction and reoxidation of the disulfide bond.

Although native beta(2)-microglobulin (beta2-m), the light chain of the major histocompatibility complex class I antigen, assumes an immunoglobulin domain fold, it is also found as a major component of dialysis-related amyloid fibrils. In the amyloid fibrils, the conformation of beta2-m is considered to be largely different from that of the native state, and a monomeric denatured form is likely to be a precursor to the amyloid fibril. To obtain insight into the conformational dynamics of beta2-m leading to the formation of amyloid fibrils, we studied the reduction and reoxidation of the disulfide bond by reduced and oxidized dithiothreitol, respectively, and the effects on the reduction of the chaperonin GroEL, a model protein that might destabilize the native state of beta2-m. We show that beta2-m occasionally unfolds into a denatured form even under physiological conditions and that this transition is promoted upon interaction with GroEL. The results imply that in vivo interactions of beta2-m with other proteins or membrane components could destabilize its native structure, thus stabilizing the amyloid precursor.

The role of disulfide bond in the amyloidogenic state of beta(2)-microglobulin studied by heteronuclear NMR.

beta(2)-Microglobulin (beta2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant beta2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced beta2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of beta2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. [(1)H]-(15)N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, DeltaCalpha, DeltaCO, and DeltaHalpha, and (3)J(HNHalpha) coupling constants indicated that both the oxidized and reduced beta2-m at pH 2.5 have marginal alpha-helical propensity at regions close to the C-terminal cysteine, although it is a beta-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of beta2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential.