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19F-NMR analysis using the optimized 19F chemical library enables the modeling of the structure of the weakly bound protein–compound complex, overcoming the difficulty in fragment-based drug discovery.

With in-cell NMR, the structural and dynamic features of proteins can be analyzed directly in living cells at atomic resolution. However, in mammalian cells, such as human cells, the structural/dynamic features and physiological functions are not fully understood at atomic resolution. In this study, we developed a method for the highly viable and efficient introduction of stable isotope-labeled proteins into human cells by electrotransfection. Thus, it was possible to perform in-cell NMR to study the structural/dynamic features of intracellular proteins with a time resolution of a minute. The transfected proteins were localized in intracellular vesicles and gradually degraded as the vesicles acidified, which could be controlled by adding EDTA, a membrane-impermeable low molecular weight compound, during electrotransfection. With this method, we could then determine the precise structure and dynamics of proteins in endosomal vesicles. Our approach demonstrates that the combination of electrotransfection and in-cell NMR provides an opportunity to study the structural/dynamic features and physiological functions of molecules taken into intact cells.

T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity., 筋萎縮性側索硬化症（ALS）の発症機構の一端を解明 --タンパク質の高密度な凝縮構造が鍵--. 京都大学プレスリリース. 2022-09-13.

Fragment-based drug discovery (FBDD), which involves small compounds

Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.

Here, we report the first synthesis of quasi-stable trimer models of full-length Aβ40 with a toxic conformation using a 1,3,5-phenyltris-l-alanyl linker at position 34, 36, or 38. The only trimer to exhibit weak neurotoxicity against SH-SY5Y cells was the one which was linked at position 38. This suggests that such a propeller-type trimer model is not prone to forming oligomers with potent neurotoxicity, which is in contrast with its corresponding dimer model.

Linear polyubiquitin chains regulate diverse signaling proteins, in which the chains adopt various conformations to recognize different target proteins. Thus, the structural plasticity of the chains plays an important role in controlling the binding events. Herein, paramagnetic NMR spectroscopy is employed to explore the conformational space sampled by linear diubiquitin, a minimal unit of linear polyubiquitin, in its free state. Rigorous analysis of the data suggests that, regarding the relative positions of the ubiquitin units, particular regions of conformational space are preferentially sampled by the molecule. By combining these results with further data collected for charge-reversal derivatives of linear diubiquitin, structural insights into the factors underlying the binding events of linear diubiquitin are obtained.

Protein persulfidation plays a role in redox signaling as an anti-oxidant. Dimers of amyloid β42 (Aβ42), which induces oxidative stress-associated neurotoxicity as a causative agent of Alzheimer's disease (AD), are minimum units of oligomers in AD pathology. Met35 can be susceptible to persulfidation through its substitution to homoCys residue under the condition of oxidative stress. In order to verify whether persulfidation has an effect in AD, herein we report a chemical approach by synthesizing disulfide dimers of Aβ42 and their evaluation of biochemical properties. A homoCys-disulfide dimer model at position 35 of Aβ42 formed a partial β-sheet structure, but its neurotoxicity was much weaker than that of the corresponding monomer. In contrast, the congener with an alkyl linker generated β-sheet-rich 8–16-mer oligomers with potent neurotoxicity. The length of protofibrils generated from the homoCys-disulfide dimer model was shorter than that of its congener with an alkyl linker. Therefore, the current data do not support the involvement of Aβ42 persulfidation in Alzheimer's disease.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

Glypican-5 (GPC5) is a heparan sulfate proteoglycan (HSPG) localized to the plasma membrane. We previously reported that in the human mesenchymal stem cell line UE6E7T3, GPC5 is overexpressed in association with transformation and promotes cell proliferation by acting as a co-receptor for Sonic hedgehog signaling. In this study, we found using an immunofluorescence microscopy that in transformed cells (U3DT), GPC5 localized not only at primary cilia on the cell surface, but also at the leading edge of migrating cells, at the intercellular bridge and blebs during cytokinesis, and in extracellular vesicles. In each subcellular region, GPC5 colocalized with the small GTPase Rab11. These observations suggest that the colocalization of GPC5 with Rab11 is crucial for its function, and that its activity in each subcellular compartment promotes proliferation of U3DT cells. Our findings indicate that GPC5 plays active and essential roles in regulation of U3DT cell proliferation, and provides several insights into the functions of GPC5 that could be elucidated by future studies.

MyD88 (myeloid differentiation factor 88) is an important protein in innate immunity. Two structural domains of MyD88 have been well characterized separately, but the global architecture of full-length MyD88 remained unclear. Here, we propose an autosuppressive mechanism of MyD88 regulated by the intramolecular interaction between the two domains.

Glypican-5 (GPC5) is a heparan sulfate proteoglycan (HSPG) localized to the plasma membrane. We previously reported that in the human mesenchymal stem cell line UE6E7T-3, GPC5 is overexpressed in association with transformation and promotes cell proliferation by acting as a co-receptor for Sonic hedgehog signaling. In this study, we found using immunofluorescence microscopy that in transformed cells (U3DT), GPC5 localized not only at primary cilia on the cell surface, but also at the leading edge of migrating cells, at the intercellular bridge and blebs during cytokinesis, and in extracellular vesicles. In each subcellular region, GPC5 colocalized with fibroblast growth factor receptor (FGFR) and the small GTPases Rab11 and ARF6, indicating that GPC5 is delivered to these regions by Rab11-associated recycling endosomes. These colocalizations suggest that GPC5 plays an important role in FGF2 stimulation of cell migration, which was abrogated by knockdown of GPC5. Our findings indicate that GPC5 plays a role in regulation of U3DT cell migration and provides several insights into the functions of GPC5 that could be elucidated by future studies.

Tensin2 (Tns2) is an essential component for the maintenance of glomerular basement membrane (GBM) structures. Tns2-deficient mice were previously shown to develop mild glomerular injury on a DBA/2 background, but not on a C57BL/6J or a 129/SvJ background, suggesting that glomerular injury by the deletion of Tns2 was strongly dependent on the genetic background. To further understand the mechanisms for the onset and the progression of glomerular injury by the deletion of Tns2, we generated Tns2-deficient mice on an FVB/N (FVB) strain, which is highly sensitive to glomerular disease. Tns2-deficient mice on FVB (FVBGN) developed severe nephrotic syndrome, and female FVBGN mice died within 8 weeks. Ultrastructural analysis revealed that FVBGN mice exhibited severe glomerular defects with mesangial process invasion of glomerular capillary tufts, lamination and thickening of the GBM and subsequent podocyte foot process effacement soon after birth. Aberrant laminin components containing α1, α2 and β1 chains, which are normally expressed in the mesangium, accumulated in the GBM of FVBGN, suggesting that these components originated from mesangial cells that invaded glomerular capillary tufts. Compared to Tns2-deficient mice on the other backgrounds in previous reports, FVBGN mice developed earlier onset of glomerular defects and rapid progression of renal failure. Thus, this study further extended our understanding of the possible genetic background effect on the deterioration of nephrotic syndrome by Tns2 deficiency.

Aggregation of 42-residue amyloid β-protein (Aβ42) can be prevented by β-sheet breaker peptides (BSBps) homologous to LVFFA residues, which are included in a β-sheet region of Aβ42 aggregates. To enhance the affinity of BSBps to the Aβ42 aggregates, we designed and synthesized β-strand-fixed peptides (BSFps) whose side chains were cross-linked by ring closing metathesis. Conformation analysis verified that the designed peptides could be fixed in β-strand conformation. Among the synthesized pentapeptides, 1 and 12, whose side chains of 2nd and 4th residues were cross-linked, significantly inhibited the aggregation of Aβ42. This suggested that β-strand-fixation of BSBps could enhance their inhibitory activity against the Aβ42 aggregation. However, pentapeptides 1 and 12 had little effect on morphology of Aβ42 aggregates (fibrils) and neurotoxicity of Aβ42 against SH-SY5Y cells.

Herein we report that a preferable inhibition of the nucleation phase of Aβ42, related to the formation of toxic oligomers, by triterpenoids from medicinal herbs originates from a salt bridge of their carboxy groups with Lys16 and 28 in Aβ42. Such a direct interaction targeting the monomer, dimer, and trimer suppressed further oligomerization. In contrast, the corresponding congeners without carboxy groups failed to do so.

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid β-protein (Aβ42), a causative agent of Alzheimer's disease (AD). Although the inhibition of Aβ42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC-MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aβ42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aβ42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. 1H-15N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aβ42 monomer. These regions form intermolecular β-sheets in Aβ42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aβ42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 to investigate whether 3 associates with Aβ42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aβ42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the β-sheet of Aβ42 nuclei, thereby suppressing further aggregation.

The physical/chemical states and properties of nanodiamonds subjected to thermal annealing and air oxidation, which are indispensable processes for the preparation of fluorescent nanodiamonds, were investigated. Specifically, the weight loss, particle size, crystal quality, chemical bonding states of carbon and oxygen, zeta potential, dispersibility, and fluorescent and optically detected magnetic resonance (ODMR) properties were determined using X-ray diffraction (XRD) analysis, transmission electron microscopy (TEM), elemental analysis, dynamic light scattering, Raman analysis, X-ray photoelectron spectroscopy (XPS), IR spectroscopy, and a home-made fluorescence and ODMR microscope. The study focused on small-sized nanodiamonds ([similar]50 nm), which are applicable for biological research. The obtained results should be useful for controlling the mutually-related physical/chemical states and properties of diamond nanoparticles.

The aggregation of the 42-residue amyloid β-protein (Aβ42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aβ42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3′,4′-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aβ42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aβ42-taxifolin adduct was suggested by mass spectrometry, Aβ42 mutants substituted at Arg5, Lys16, and/or Lys28 with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aβ42 mutants at Lys16 and/or Lys28 except for the mutant at Arg5. In addition, the aggregation of Aβ42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aβ42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aβ42 as well as Aβ42. Furthermore, interaction of (+)-taxifolin with the β-sheet region in Aβ42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aβ42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aβ42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy. Background: The inhibitory mechanism of Aβ42 aggregation by flavonoid is fully unknown. Results: The oxidant enhanced the inhibitory activity of (+)-taxifolin against Aβ42 aggregation by forming Aβ42-taxifolin adducts between the Lys residues and oxidized (+)-taxifolin. Conclusion: The inhibitory activity of catechol-type flavonoids requires autoxidation to form an o-quinone to react with Lys. Significance: These may help design promising inhibitors against Aβ42 aggregation for Alzheimer disease therapy.

Silymarin, the seed extract of Silybium marianum, has preventive effects against Alzheimer's disease-like pathogenesis in vivo. We isolated (+)-taxifolin (4) from silymarin as an inhibitor of aggregation of the 42-residue amyloid β-protein. Structure-activity relationship studies revealed the 3',4'-dihydroxyl groups to be critical to the anti-aggregative ability, whereas the 7-hydroxyl group and the stereochemistry at positions 2 and 3 were not important.

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBDChiA1) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBDChiA1 were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBDChiA1. Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBDChiA1 in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

This research was originally published in Journal of Biological Chemistry. Hiroaki Tanaka, Ken-ichi Akagi, Chitose Oneyama, Masakazu Tanaka, Yuichi Sasaki, Takashi Kanou, Young-Ho Lee, Daisuke Yokogawa, Marc-Werner Dobenecker, Atsushi Nakagawa, Masato Okada and Takahisa Ikegami. Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains. Journal of Biological Chemistry. 2013; 288, 15240-15254. © the American Society for Biochemistry and Molecular Biology., Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βCloop of the SH2 domain.

Amyloid β-protein (Aβ42) oligomerization is an early event in Alzheimer's disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aβ42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aβ42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aβ42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aβ42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aβ42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aβ42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy., アルツハイマー病特有のアミロイドβ立体構造に特異的な抗体の開発―より正確な診断手法への応用に期待―. 京都大学プレスリリース. 2016-07-06.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation.

Amyloid fibrils in senile plaque mainly consist of the 40-mer and 42-mer amyloid β-proteins (Aβ40 and Aβ42). Although Aβ42 plays more important role in the pathogenesis of Alzheimer's disease (AD), Aβ40 could be involved in the progression of AD pathology because of its large amount. Recent studies revealed that variable sizes of Aβ oligomers contributed to the neuronal death and cognitive dysfunction. However, how large oligomeric species are responsible for AD pathogenesis remains unclear. We previously proposed a toxic dimer model of Aβ with turn structure at positions 22 and 23 using solid-state NMR and systematic proline replacement. Based on this model, we herein show the synthesis and biological activities of an E22P-Aβ40 dimer at position 30, which was connected to l, l-2, 6-diaminopimeric acid. The E22P-Aβ40 dimer formed stable 6∼8-mer oligomers without amyloid fibrils, but was not neurotoxic on human neuroblastoma cells. On the other hand, E22P-Aβ40 generated high molecular-weight oligomers into fibrils, and showed the neurotoxicity. These results suggest that such kind of Aβ40 dimer with a parallel β-sheet might not be pathological.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).

Chitinase C from Streptomyces griseus HUT6037 was discovered as the first bacterial chitinase in family 19 other than chitinases found in higher plants. Chitinase C comprises two domains: a chitin-binding domain (ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain for digesting chitin. The structure of ChBD(ChiC) was determined by means of 13C-, 15N-, and 1H-resonance nuclear magnetic resonance (NMR) spectroscopy. The conformation of its backbone comprised two beta-sheets composed of two and three antiparallel beta-strands, respectively, this being very similar to the backbone conformations of the cellulose-binding domain of endoglucanase Z from Erwinia chrysanthemi (CBD(EGZ)) and the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 (ChBD(ChiA1)). The interaction between ChBD(ChiC) and hexa-N-acetyl-chitohexaose was monitored through chemical shift perturbations, which showed that ChBD(ChiC) interacted with the substrate through two aromatic rings exposed to the solvent as CBD(EGZ) interacts with cellulose through three characteristic aromatic rings. Comparison of the conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin- and cellulose-binding domains, which have three solvent-exposed aromatic residues responsible for binding to polysaccharides, has suggested that they have adopted versatile binding site conformations depending on the substrates, with almost the same backbone conformations being retained.

Alzheimer’s disease (AD) is caused by abnormal deposition (fibrillation) of a 42-residue amyloid β-protein (Aβ42) in the brain. During the process of fibrillation, the Aβ42 takes the form of protofibrils with strong neurotoxicity, and is thus believed to play a crucial role in the pathogenesis of AD. To elucidate the supramolecular structure of the Aβ42 protofibrils, the intermolecular proximity of the Ala-21 residues in the Aβ42 protofibrils was analyzed by 13C–13C rotational resonance experiments in the solid state. Unlike the Aβ42 fibrils, an intermolecular 13C–13C correlation was not found in the Aβ42 protofibrils. This result suggests that the β-strands of the Aβ42 protofibrils are not in an in-register parallel orientation. Aβ42 monomers would assemble to form protofibrils with the β-strand conformation, then transform into fibrils by forming intermolecular parallel β-sheets.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.

Ubiquitin is a post-translational modifier that is involved in cellular functions through its covalent attachment to target proteins. Ubiquitin can also be conjugated to itself at seven lysine residues and at its amino terminus to form eight linkage-specific polyubiquitin chains for individual cellular processes. The Lys63-linked polyubiquitin chain is recognized by tandem ubiquitin-interacting motifs (tUIMs) of Rap80 for the regulation of DNA repair. To understand the recognition mechanism between the Lys63-linked diubiquitin (K63-Ub(2)) and the tUIMs in solution, we determined the solution structure of the K63-Ub(2):tUIMs complex by using NOE restraints and RDC data derived from NMR spectroscopy. The structure showed that the tUIMs adopts a nearly straight and single continuous α-helix, and the two ubiquitin units of the K63-Ub(2) separately bind to each UIM motif. The interfaces are formed between Ile44-centered patches of the two ubiquitin units and the hydrophobic residues of the tUIMs. We also showed that the linker region between the two UIM motifs possesses a random-coil conformation in the free state, but undergoes the coil-to-helix transition upon complex formation, which simultaneously fixes the relative position of ubiquitin subunits. These data suggest that the relative position of ubiquitin subunits in the K63-Ub(2):tUIMs complex is essential for linkage-specific binding of Rap80 tUIMs.

Aggregation of 42-residue amyloid β-protein (Aβ42) plays a pivotal role in the etiology of Alzheimer’s disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of Aβ42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the Aβ42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the Aβ42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the Aβ42 fibrils. The observed 13C–13C cross peaks indicated that curcumin interacts with the 12th and 17–21st residues included in the β-sheet structure in the Aβ42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the Aβ42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the Aβ42 fibrils.

Compact tension(CT) tests were performed at various test speeds on epoxy resin specimens with different hardness achieved by varying base compounds ratio. The calculated toughness, Kqi, for unstable crack initiation, was compared with Charpy impact values, and the correlation between the two was discussed with the characteristics of the load displacement curve and the fracture appearance.The experiments showed that the toughness value, Kqi, depended strongly on the test speed, and was not correlated with impact value, or with static strength. However, there was a linear relation between the toughness and impact values for the specimens where the load-deflection curve and fracture appearance indicated an initial small slow-stable crack followed by rapid-unstable fracture. This occurred in very slow CT tests, where appearance of the rapid fracture was similar to that in impact specimens.In other cases, where the fracture was slow-stable, stick slip type rapid-unstable or rapid stable, Kqi was independent of the impact value, and the fracture appearance was quite different. The Kqa values, for crack arrest, were not correlated with impact values. It must be noted that the toughness values of CT tests should only be compared when the fracture types are similar.

Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.