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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.

Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.

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 a physicochemical approach that combined thermodynamic studies with structural analyses in solution, to clarify the binding mechanism. First, using a combination of CD, NMR, 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, using isothermal titration calorimetry (ITC), we revealed that the interaction of Mint3 and FIH-1 produces an enormous change in enthalpy and entropy. 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 78-88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part to the interaction, where one contributes to the affinity (ΔG), while the other only affects enthalpy (ΔH), by forming non-covalent bonds. This insight can serve as a foothold for further investigation of IDRs and drug development for cancer therapy.

An increasing number of proteins, which have neither regular secondary nor well-defined tertiary structures, have been found to be present in cells. The structure of these proteins is highly flexible and disordered under physiological (native) conditions, and they are called “intrinsically disordered” proteins (IDPs). Many of the IDPs are involved in interactions with other biomolecules such as DNA, RNA, carbohydrates, and proteins. While these IDPs are largely unstructured by themselves, marked conformational changes often occur upon binding to an interacting partner, which is known as the “coupled folding and binding mechanism”, which enable them to change the conformation to become compatible with the shape of the multiple target biomolecules. We have studied the structure and interaction of eukaryotic transcription factors Sp1 and TAF4, and found that both of them have long intrinsically disordered regions (IDRs). One of the IDRs in Sp1 exhibited homo-oligomer formation. In addition, the same region was used for the interaction with another IDR found in the TAF4 molecule. In both cases, we have not detected any significant conformational change in that region, suggesting a prominent and novel binding mode for IDPs/IDRs, which are not categorized by the well-accepted concept of the coupled folding and binding mechanism.

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 β/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.

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.

The expression of eukaryotic genes is precisely controlled by interactions between general transcriptional factors and promoter-specific transcriptional activators. The fourth element of TATA-box binding protein-associated factor (TAF4), an essential subunit of the general transcription factor TFIID, serves as a coactivator for various promoter-specific transcriptional regulators. Interactions between TAF4 and site-specific transcriptional activators, such as Sp1, are important for regulating the expression levels of genes of interest. However, only limited information is available on the molecular mechanisms underlying the interactions between these transcriptional regulatory proteins. We herein analyzed the interaction between the transcriptional factors Sp1 and TAF4 using high-resolution solution nuclear magnetic resonance spectroscopy. We found that four glutamine-rich (Q-rich) regions in TAF4 were largely disordered under nearly physiological conditions. Among them, the first Q-rich region in TAF4 was essential for the interaction with another Q-rich region in the Sp1 molecule, most of which was largely disordered. The residues responsible for this interaction were specific and highly localized in a defined region within a range of 20-30 residues. Nevertheless, a detailed analysis of 13 C-chemical shift values suggested that no significant conformational change occurred upon binding. These results indicate a prominent and exceptional binding mode for intrinsically disordered proteins other than the well-accepted concept of "coupled folding and binding."

The abnormal aggregation of amyloid β-peptide (Aβ) is central to the pathogenesis of Alzheimer's disease, the major form of dementia. Aromatic π-π interactions have been suggested to play a crucial role in the aggregation of not only Aβ, but also other amyloidogenic proteins. In this study, each or all phenylalanine (Phe) residues at the 4th, 19th, and 20th positions of Aβ-(1-40) were substituted by hydrophobic cyclohexylalanine (Cha), which is sterically similar to Phe, but lacks π-electrons, to reveal effects of interactions involving π-electrons on the aggregation of Aβ both in aqueous solution and GM1-containing membranes. We found that each Cha substitution significantly inhibited fibril formation by Aβ, indicating a pivotal role of aromatic interactions. Furthermore, the Aβ analog with three Cha residues effectively retarded the fibrillation of the wild-type Aβ.

The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene-specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter-specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP-associated factor TAF4. We herein report the structural details of the glutamine-rich domains (Q-domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q-domains of Sp1 and four Q-domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q-domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well-accepted concept of "coupled folding and binding."

The abnormal aggregation of amyloid β-protein (Aβ) 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 Aβ-(1-40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel β-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 β-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aβ is included in the β-sheet. Chemical cross-linking experiments using Cys-substituted Aβs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel β-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.

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.

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. The extracellular matrix glycoprotein tenascin-C stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation and suppresses pro-inflammatory cytokines.

Here I review the molecular mechanisms by which water-soluble monomeric amyloid-β (Aβ) peptides are transformed into well-organized supramolecular complexes called amyloid fibrils. The mechanism of amyloid formation is considered theoretically on the basis of experimental results, and the structural and mechanistic similarities of amyloid fibrils to three-dimensional crystals are highlighted. A number of important results from the literature are described. These include the observation that a correct ratio of monomer association and dissociation rate constants is key for formation of well-organized amyloid fibrils. The dynamic nature of the amyloid-β structure is discussed, along with the possibly obligate requirement of the transient formation of a hairpin-like fold prior to its incorporation into amyloid fibrils. Many rounds of monomer association and dissociation events may be present during an apparently silent lag-period. Amongst these association/dissociation events, interaction between the C-terminal regions of the Aβ peptide seems to be more favored. Such association and dissociation events occurring in a “trial-and-error” fashion may be an important requirement for the formation of well-organized amyloid fibrils.

The conversion of soluble, nontoxic amyloid β-proteins (Aβ) to aggregated, toxic forms rich in β-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 Aβ. Our group has previously reported that amyloid fibrils of Aβ 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 β-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 Aβ-(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. Aβ-(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 β-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 Aβ with an antiparallel β-sheet structure by providing less polar environments.

The promoter-specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N-terminal half of the protein are characterized by glutamine-rich (Q-rich) sequences. These Q-rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA-binding protein associated factors. Furthermore, the self-association of Sp1 via Q-rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a “distal” GC-box synergistically interacts with another Sp1 molecule at a “proximal” binding site. Although the formation of multimers via Q-rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q-rich domains. We analyzed the structural details of isolated glutamine-rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C-terminal half was responsible for the self-association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self-association between QB domains would be responsible, at least in part, for the formation of multimers by full-length Sp1 molecules that has been proposed to occur during transcriptional activation.

A structure-activity relationship study on a highly potent CXC chemokine receptor type 4 (CXCR4) antagonist, FC131 [cyclo(-d-Tyr(1)-Arg(2)-Arg(3)-Nal(4)-Gly(5)-)], was carried out using a series of alkene isosteres of the d-Tyr(1)-l/d-Arg(2) dipeptide to investigate the binding mode of FC131 and its derivatives with CXCR4. The structure-activity relationships of isostere-containing FC131 analogues were similar to those of the parent FC131 and its derivatives, suggesting that a trans-conformer of the d-Tyr(1)-Arg(2) peptide bond is the dominant contributor to the bioactive conformations of FC131. Although NMR analysis demonstrated that the two conformations of the peptidomimetic containing the d-Tyr(1)-d-Arg(2) isostere are possible, binding-mode prediction indicated that the orientations of the alkene motif within d-Tyr(1)-MeArg(2) peptidomimetics depend on the chirality of Arg(2) and the β-methyl group of the isostere unit, which makes the dominant contribution for binding to the receptor. The most potent FC122 [cyclo(-d-Tyr(1)-d-MeArg(2)-Arg(3)-Nal(4)-Gly(5)-)] bound with CXCR4 by a binding mode different from that of FC131.

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ∼0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ∼0.013 and ∼0.044), the helical species and aggregated β-sheets (∼15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ∼0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.

Conversion of the soluble, non-toxic amyloid β-protein (Aβ) into an aggregated, toxic form rich in β-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 Aβ-(1-40) specifically bound to a ganglioside cluster forms cytotoxic fibrils via a conformational transition from an α-helix-rich structure to a β-sheet-rich one. In the present study, we compared the interaction of Aβ-(1-40) and Aβ-(1-42) with both model and living cell membranes. Aβ-(1-42) exhibited lipid specificity and affinity similar to Aβ-(1-40), though its amyloidogenic activity was more than 10-fold that of Aβ-(1-40). Antibody staining experiments, using the A11 antibody specific to Aβ 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. Aβ-(1-40) formed long fibrils extruding to the aqueous phase, whereas Aβ-(1-42) fibrils appeared to be laterally co-assembled and short.

The conversion of the soluble, nontoxic amyloid-beta (Abeta) peptide into an aggregated, toxic form rich in beta-sheets is considered a key step in the development of Alzheimer's disease. Whereas growing evidence indicates that the Abeta amyloid fibrils consist of in-register parallel beta-sheets, little is known about the structure of soluble oligomeric intermediates because of their transient nature. To understand the mechanism by which amyloid fibrils form, especially the initial development of the "nucleus" oligomeric intermediates, we prepared covalently linked dimeric Abeta peptides and analyzed the kinetics of the fibril-forming process. A covalent bond introduced between two Abeta molecules dramatically facilitated the spontaneous formation of aggregates with a beta-sheet structure and affinity for thioflavin T. Transmission electron microscopy revealed, however, that these aggregates differed in morphology from amyloid fibrils, more closely resembling protofibrils. The protofibril-like aggregates were not the most thermodynamically stable state but were a kinetically trapped state. The results emphasize the importance of the conformational flexibility of the Abeta molecule and a balance in the association and dissociation rate for the formation of rigid amyloid fibrils.

A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR.

To clarify the mechanism of interaction between chaperonin GroEL and substrate proteins, we studied the conformational changes; of the fifth domain of human beta(2)-glycoprotein I upon binding to GroEL. The fifth domain has a large flexible loop, containing several hydrophobic residues surrounded by positively charged residues, which has been proposed to be responsible for the binding of beta(2)-glycoprotein I to negatively charged phospholipid membranes. The reduction by dithiothreitol of the three intramolecular disulfide bonds of the fifth domain was accelerated in the presence of stoichiometric amounts of GroEL, indicating that the fifth domain was destabilized upon interaction with GroEL. To clarify the GroEL-induced destabilization at the atomic level, we performed H/(2)H exchange of amide protons using heteronuclear NMR spectroscopy. The presence of GroEL promoted the H/(2)H exchange of most of the protected amide protons, suggesting that, although the flexible loop of the fifth domain is likely to be responsible for the initiation of binding to GroEL, the interaction with GroEL destabilizes the overall conformation of the fifth domain.

This research was originally published in the Journal of Biological Chemistry. Young-Ho Lee, Kosuke Tamura, Masahiro Maeda, Masaru Hoshino, Kazumasa Sakurai, Satoshi Takahashi, Takahisa Ikegami, Toshiharu Hase, and Yuji Goto. Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange. J. Biol. Chem. 2007; 282, 5959-5967. © the American Society for Biochemistry and Molecular Biology, NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP+ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP+)-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pDr values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pDr values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP+-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP+-binding domain at pDr 8.0, the daytime pH in chloroplasts, than at pDr 6.0 is likely to be involved in the increased binding of NADP+ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

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.

Interaction of ×-Lactoglobulin with Chaperonin Groel

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.

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.

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., Ubiquitin is a stable and soluble protein, but it is commonly found in inclusion bodies in neurodegenerative disorders and cancer. Here, Morimoto et al. report that increasing ubiquitin chain length leads to the formation of amyloid-like fibrils, which are degraded by an autophagy mechanism.

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of β2-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid β-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded β-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.

The recombinant V(L) domain that represents the variable part of the light chain (type kappa) of mouse monoclonal antibody F11 directed against human spleen ferritin was found to form amyloid fibrils at acidic pH as evidenced by electron microscopy, thioflavin T binding, and apple-green birefringence after Congo red staining. This is the first demonstration of amyloid fibril formation of the mouse V(L) domain. To understand the mechanism of acidic pH-induced amyloid fibril formation, conformational changes of the V(L) domain were studied by one-dimensional NMR, differential scanning calorimetry, analytical ultracentrifugation, hydrophobic dye binding, far-UV circular dichroism, and tryptophan fluorescence. The results indicated accumulation of two intermediate states during acid unfolding, which might be responsible for amyloid fibril formation. The more structured intermediate that exhibited maximal accumulation at pH 3 retained the nativelike secondary structure and a hydrophobic core, but exposed hydrophobic surfaces that bind 8-anilino-1-naphthalenesulfonate. Below pH 2, a more disordered intermediate with dequenched tryptophan fluorescence but still retaining the beta-sheet structure accumulated. The optimal pH of amyloid fibril formation (i.e., pH 4) was close to the optimal pH of the accumulation of the nativelike intermediate, suggesting that the amyloid fibrils might be formed through this intermediate.

The abnormal deposition of amyloids by amyloid-β protein (Aβ) is a pathological hallmark of Alzheimer's disease (AD). Aged rodents rarely develop the characteristic lesions of the disease, which is different from the case in humans. Rodent Aβ (rAβ) differs from human Aβ (hAβ) only in the three substitutions of Arg to Gly, Tyr to Phe, and His to Arg at positions 5, 10, and 13, respectively. Understanding the reason why rodent Aβ does not form amyloids is important to revealing factors that cause the abnormal aggregation of Aβ under pathologic conditions. We have proposed that the binding of Aβ to membranes with ganglioside clusters plays an important role in the abnormal aggregation of Aβ. In this study, we compared hAβ and rAβ in terms of aggregation on neuronal cells, on raftlike model membranes, and in buffer. We found that rAβ formed amyloid fibrils similar to those of hAβ in buffer solution. In contrast, on cell membranes and raftlike membranes, hAβ formed toxic, mature amyloid fibrils, whereas rAβ produced less toxic protofibrils that were not stained by the amyloid-specific dye Congo red. Thus, our ganglioside cluster-mediated amyloidogenesis hypothesis explains the immunity of rodents from cerebral Aβ amyloid deposition, strengthening the importance of ganglioside clusters as a platform of abnormal Aβ deposition in the pathology of AD.

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'-(β,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.

beta(2)-Glycoprotein I (beta(2)-GPI) is a plasma protein that binds to negatively charged substances such as DNA, heparin, and anionic phospholipids. The interaction of beta(2)-GPI with anionic phospholipids is intriguing in the context of the autoimmune disease antiphospholipid syndrome. To extend understanding of the binding mechanism to phospholipids, the interactions of beta(2)-GPI with amphiphiles, i.e., sodium lauryl sulfate and lysophospholipids, were examined. These amphiphiles induced the aggregation of beta(2)-GPI below the critical micelle concentration, indicating that the interaction of beta(2)-GPI with monodispersed amphiphiles is unstable, resulting in the formation of large aggregates. However, highly soluble monocaproylphosphatidic acid did not induce aggregation, suggesting that the hydrophobicity of the acyl chain is also an important factor for aggregate formation in addition to negative charges in the headgroup. A series of experiments using deletion mutants and a peptide showed that the fifth domain of beta(2)-GPI (domain V) is responsible for formation of aggregates observed for intact full-length beta(2)-GPI. In addition, the flexible loop (F307-C326) in the C-terminal of domain V, which consists of hydrophobic and positively charged residues, was identified as the important region for aggregation. These results indicate that beta(2)-GPI binds to the amphiphiles through the flexible loop of domain V, resulting in formation of large aggregates where both electrostatic and hydrophobic interactions are involved.

We have defined the structural and dynamic properties of an early folding intermediate of β-lactoglobulin known to contain non-native α-helical structure. The folding of β-lactoglobulin was monitored over the 100 μs–10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 μs is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising β-strands F, G and H, and the main α-helix, which was thus identified as the folding core of β-lactoglobulin. At the same time, weak protection (up to ∼10-fold) of amide protons in a segment spanning residues 12–21 is consistent with formation of marginally stable non-native α-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like α/β core domain.

Among various alcohols, those substituted with fluorine, such as 2,2,2-trifluoroethanol (TFE) or 3,3,3,3‘,3‘,3‘-hexafluoro-2-propanol (HFIP), have a marked potential to induce the formation of α-helical structures in peptides and to denature the native structures of proteins. However, the mechanism by which these alcohols exert their effects is unknown. Melittin, a bee venom peptide, is unfolded in the absence of alcohol, but is transformed to an α-helical structure upon addition of alcohols. On the other hand, addition of alcohols to β-lactoglobulin, a predominantly β-sheet protein, denatures the molecule and transforms it to an α-helical structure. We examined the role of several factors in these alcohol-induced transitions, i.e., relative dielectric constant, strength of hydrogen bond estimated by the pH titration of salicylic acid, and clustering of alcohol molecules measured by solution X-ray scattering. Although relative dielectric constant and hydrogen bond strength were confirmed to be important, ...

Whereas bovine β-lactoglobulin is a predominantly β-sheet protein, it has a marked α-helical preference and can be considered to be a useful model of the α → β transition, a key issue for understanding the folding and biological function of a number of proteins. In order to understand the mechanism of the α → β transition, the backbone structures of the recombinant bovine β-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol were characterized by 1H, 13C and 15N multidimensional NMR spectroscopy. Overall, the secondary structures in the native state were similar to those of the crystal structure. On the other hand, β-lactoglobulin in the 2,2,2-trifluoroethanol state was composed of many α-helical segments. The presence of the persistent α-helices in the helical state and the core β-sheet in the native state suggested that during folding native-like core β-sheet and several non-native helices are formed first and the remaining β-sheet is subsequently “induced” through interaction with the pre-existing β-sheet.

The formation of neurotoxic aggregates by amyloid-β protein (Aβ) is considered to be a key step in the onset of Alzheimer's disease. Although cytotoxicity of various Aβ assemblies formed in aqueous solution has been extensively investigated, the relationship between the aggregate size and cytotoxicity is elusive for the membrane-mediated aggregation process [1]. In this study, the aggregational processes of Aβ-(1−42) and Aβ-(1−40) on living SH-SY5Y neuroblastoma cells were monitored by fluorescence correlation spectroscopy and the amyloid-specific dye Congo red. At the same time, cytotoxic events were studied for the more toxic Aβ-(1−42). Both Aβ-(1−42) and Aβ-(1−40) formed amyloids after forming preamyloid aggregates composed of ∼15 Aβ molecules in agreement with the liposomal study [2]. The formation of amyloids was needed to induce DNA fragmentation (a late apoptotic event) and a reduction in cell proliferation ability. At this stage the translocation of NFκB from the cytosol to the nucleus was observed. In contrast, smaller oligomers triggered the exposure of phosphatidylserine, an early apoptotic process. However, this event did not reduce cell proliferation ability. Thus, amyloid formation is a key event in Aβ-induced cytotoxicity.[1] K. Matsuzaki, Acc. Chem. Res. 47, 2397 (2014).[2] K. Ikeda et al., Biochemistry 50, 6433 (2011).

The most common isoforms of amyloid-β (Aβ) proteins are composed of 40 or 42 amino acid residues. While Aβ-(1–40) is the predominant species, Aβ-(1–42) is more fibrillogenic and neurotoxic, suggesting that Aβ-(1–42) plays a critical role in the initiation of amyloid fibril formation. We investigated the mechanisms by which soluble Aβ-(1–40) associates with preformed Aβ-(1–42) seeds. A paramagnetic relaxation enhancement analysis showed that the Aβ-(1–40) monomer and Aβ-(1–42) seed interact via their C-terminal region in a parallel fashion, and the N-terminal part does not to contribute to the interaction. Structured summary of protein interactions A beta-(1–40) and A beta-(1–42) bind by fluorescence technology ( View interaction ) A beta-(1–42) and A beta-(1–40) bind by nuclear magnetic resonance ( View interaction )

The promoter-specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N-terminal half of the protein are characterized by glutamine-rich (Q-rich) sequences. These Q-rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA-binding protein associated factors. Furthermore, the self-association of Sp1 via Q-rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a “distal” GC-box synergistically interacts with another Sp1 molecule at a “proximal” binding site. Although the formation of multimers via Q-rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q-rich domains. We analyzed the structural details of isolated glutamine-rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C-terminal half was responsible for the self-association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self-association between QB domains would be responsible, at least in part, for the formation of multimers by full-length Sp1 molecules that has been proposed to occur during transcriptional activation.

Mastoparan, a basic tetradecapeptide from wasp venom, has been considered to be unfolded under aqueous conditions. On the basis of the far-UV circular dichroism spectrum, we found that sodium perchlorate at molar concentrations stabilizes an alpha-helical structure of mastoparan. To understand the mechanism of the perchlorate-induced stabilization of the alpha-helical structure, we synthesized a dimeric form of mastoparan derivative, which was linked at the C terminal by a disulfide bond. The linkage decreased the concentration of perchlorate required to stabilize the alpha-helical structure by 30-fold. With the dimeric mastoparan derivative, we measured the effects of several salts such as sodium trichloroacetate, sodium trifluoroacetate, and sodium chloride. The concentration of salts required to induce the conformational transition varied and the order of effectiveness of different salts was consistent with the electroselectivity series of anions toward anion-exchange resins, indicating that the anion binding to the positively charged amino groups is responsible for the transition. These results suggest that the salt-induced formation of the alpha-helical state of mastoparan can be explained by a mechanism similar to that proposed for the salt-induced conformational transition of melittin.

Whereas the salt-dependent conformational transition of acid-denatured horse ferricytochrome c at pH 2 is approximated by a two-state mechanism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 591-596], the corresponding transition in D2O has been proposed to involve a noncompact, alpha-helical intermediate state (the pre-molten globule state) [Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1045-1061]. To examine the proposed difference in the conformational transitions, we carried out the HCl and DCl titrations of cytochrome c in H2O and D2O, respectively, measured by far-UV circular dichroism, tryptophan fluorescence, and Soret absorption. In both D2O and H2O, unfolding from the native state to the acid-unfolded state and subsequent refolding to the molten globule state were observed. In either solvent, the conformational transitions were well approximated by a minimal three-state mechanism consisting of the native, molten globule, and acid-unfolded states. Thus, our results did not substantiate the presence of a pre-molten globule state in D2O. Acetylation of amino groups of cytochrome c is known to stabilize the molten globule state at pH 2. On the basis of the three-state mechanism, we constructed a conformational phase diagram for the effect of pH and the degree of acetylation. This phase diagram was similar to that of the pH- and salt-dependent conformational transition of cytochrome c, suggesting that the effects of acetylation on the conformational states are similar to those of salt.