Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276.

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro.

Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation (LLPS) in the absence of PrPSc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by ß-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid-solid phase transition could be the intermediate steps in the conversion of prions.

Short disordered protein segment regulates cross-species transmission of a yeast prion.

Soluble prion proteins contingently encounter foreign prion aggregates, leading to cross-species prion transmission. However, how its efficiency is regulated by structural fluctuation of the host soluble prion protein remains unsolved. In the present study, through the use of two distantly related yeast prion Sup35 proteins, we found that a specific conformation of a short disordered segment governs interspecies prion transmissibility. Using a multidisciplinary approach including high-resolution NMR and molecular dynamics simulation, we identified critical residues within this segment that allow interspecies prion transmission in vitro and in vivo, by locally altering dynamics and conformation of soluble prion proteins. Remarkably, subtle conformational differences caused by a methylene group between asparagine and glutamine sufficed to change the short segment structure and substantially modulate the cross-seeding activity. Thus, our findings uncover how conformational dynamics of the short segment in the host prion protein impacts cross-species prion transmission. More broadly, our study provides mechanistic insights into cross-seeding between heterologous proteins.

Development of flavonoid probes and the binding mode of the target protein and quercetin derivatives.

This study investigated the mechanism underlying anti-cancer cell migration activity of quercetin derivatives by investigating the binding mode of the target protein. Five flavonoid probes were newly synthesized, and pull down assay using synthesized flavonoid probes indicated matrix metalloproteinase-1 (MMP-1) as the target protein of quercetin derivatives. Quercetin and 3-O-methylquercetin (3MQ) inhibited MMP-1. SPR analysis demonstrated dose dependent interaction between quercetin derivatives and recombinant MMP-1 catalytic domain. And 1H-15N heteronuclear single quantum coherence (HSQC) NMR analysis using 15N-labeled MMP-1 catalytic domain indicated that 3MQ interacted around metal ions in the MMP-1. The development of flavonoid probes can broaden the possibility to discover the new target proteins and elucidate the core mechanisms of the multi bioactivity of flavonoids.

Comparing microRNA in milk small extracellular vesicles among healthy cattle and cattle at high risk for bovine leukemia virus transmission.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by bovine leukemia virus (BLV) infection. In Japan, cattle diagnosed with EBL are not permitted for human consumption by the law, thereby causing serious economic losses to farmers. The prevalence of BLV is high in Japan (40.9% in dairy cattle and 28.7% in beef cattle, respectively), which makes it difficult to perform the test-and-slaughter of BLV-infected cattle. This necessitates preventing the spread of BLV infection in cattle by early detection, segregation, and the removal of BLV-infected cattle with high proviral load, which are considered high risk for BLV transmission. We aimed to identify cattle that were at high risk for BLV transmission by comparing microRNA (miRNA) profiles in milk small extracellular vesicles (sEV). At first, miRNA profiles in sEV were compared among 4 uninfected cattle and 4 BLV-infected cattle with high proviral load by using a microarray containing mixed probes for miRNA of cattle and humans. Significantly lower amounts of hsa-miR-557 and hsa-miR-19b-1-5p, and insignificantly but higher amounts of hsa-miR-424-5p were observed in milk sEV from BLV-infected cattle than those from uninfected cattle. Next, to evaluate the utility of the aforementioned miRNAs for the identification of cattle that were at high risk for BLV transmission, we performed quantitative real-time PCR using milk sEV newly collected from 5 uninfected cattle and 17 BLV-infected cattle with high proviral load. The cycle threshold value of hsa-miR-424-5p was significantly lower in milk sEV from BLV-infected cattle. The PCR detection was unavailable or a significant difference was not observed for hsa-miR-557 and hsa-miR-19b-1-5p, respectively. These results suggest that the amount of hsa-miR-424-5p was higher in milk sEV from BLV-infected cattle and increasing the hsa-miR-424-5p in milk sEV could be one of the characteristic trends in cattle that are high risk for BLV transmission. Moreover, assessing characteristic miRNA amounts in milk sEV, which can be recovered twice a day by milking, could be useful for the routine monitoring of cattle in dairy herds instead of blood collection.

Characterization of miRNAs in Milk Small Extracellular Vesicles from Enzootic Bovine Leukosis Cattle.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs-bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p-which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans-were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.

Molecular Mechanisms of Aggregation of Canine SOD1 E40K Amyloidogenic Mutant Protein.

Canine degenerative myelopathy (DM) is a human amyotrophic lateral sclerosis (ALS)-like neurodegenerative disease. It is a unique, naturally occurring animal model of human ALS. Canine DM is associated with the aggregation of canine superoxide dismutase 1 (cSOD1), which is similar to human ALS. Almost 100% of cases in dogs are familial, and the E40K mutation in cSOD1 is a major causative mutation of DM. Therefore, it is important to understand the molecular mechanisms underlying cSOD1(E40K) aggregation. To address this, we first analyzed the structural model of wild type cSOD1. Interactions were evident between amino acid E40 and K91. Therefore, the mutation at residue E40 causes loss of the interaction and may destabilize the native structure of cSOD1. Differential scanning fluorimetry revealed that the E40K mutant was less stable than the wild type. Moreover, stability could be recovered by the E40K and K91E double mutation. Acceleration of amyloid fibril formation in vitro and aggregate formation in cells of cSOD1(E40K) was also suppressed by the introduction of this double mutation in thioflavin T fluorescence assay results and in transfectant cells, respectively. These results clearly show the importance of the interaction between amino acid residues E40 and K91 in cSOD1 for the stability of the native structure and aggregation.

Single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation.

The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed in Escherichia coli in the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for "structural screening" of refolded proteins in the case that the "functional screening" like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.

Molecular basis for diversification of yeast prion strain conformation.

Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Kaempferol Has Potent Protective and Antifibrillogenic Effects for α-Synuclein Neurotoxicity In Vitro.

Aggregation of α-synuclein (α-Syn) is implicated in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Therefore, the removal of α-Syn aggregation could lead to the development of many new therapeutic agents for neurodegenerative diseases. In the present study, we succeeded in generating a new α-Syn stably expressing cell line using a piggyBac transposon system to investigate the neuroprotective effect of the flavonoid kaempferol on α-Syn toxicity. We found that kaempferol provided significant protection against α-Syn-related neurotoxicity. Furthermore, kaempferol induced autophagy through an increase in the biogenesis of lysosomes by inducing the expression of transcription factor EB and reducing the accumulation of α-Syn; thus, kaempferol prevented neuronal cell death. Moreover, kaempferol directly blocked the amyloid fibril formation of α-Syn. These results support the therapeutic potential of kaempferol in diseases such as synucleinopathies that are characterized by α-Syn aggregates.

Structural and functional evaluation of single-chain Fv antibody HyC1 recognizing the residual native structure of hen egg lysozyme.

Evaluation of the molecular mechanisms by which an antibody recognizes a specific antigen could help in better understanding of the protein recognition mechanisms. We previously showed that anti-hen egg lysozyme (HEL) monoclonal antibody, HyC1, recognized the structural and hydrodynamic change in HEL. Here, we generated HyC1 single-chain Fv (scFv), and characterized it using different structural and biophysical methods. Similar to HyC1 monoclonal antibody, HyC1 scFv could recognize native HEL from carboxymethylated Cys6 and Cys127 HEL (CM6,127-HEL). Comparison of the binding thermodynamics of HyC1 scFv between HEL and CM6,127-HEL showed that the binding enthalpy change was different, while the binding entropy was remained unchanged. The results indicated that the fluctuation of the residual native structure in both HEL and CM6,127-HEL was similar. The NMR experiments for 15N-labeled HyC1 scFv indicated that the flexibility of HyC1 scFv decreased upon the binding to HEL.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

Cavities and Excited States in Proteins.

Protein cavities or voids are observed as defects in atomic packing. Cavities have long been suggested to play important roles in protein dynamics and function, but the underlying origin and mechanism remains elusive. Here, recent studies about the cavities characterized by high-pressure NMR spectroscopy have been reviewed. Analysis of the pressure-dependent chemical shifts showed both linear and nonlinear response of proteins to pressure. The linear response corresponded to compression within the native ensemble, while the nonlinear response indicated the involvement of low-lying excited states that were different from the native state. The finding of non-linear pressure shifts in various proteins suggested that the existence of the low-lying excited states was common for globular proteins. However, the absolute nonlinear coefficient values varied significantly from protein to protein, and showed a good correlation with the density of cavities. Extensive studies on hen lysozyme as a model system showed that the cavity hydration and water penetration into the interior of proteins was an origin of the conformational transition to the excited states. The importance of cavities for protein function and evolution has also been explained. In addition to these "equilibrium" cavities, there are also "transient" cavities formed in the interior of the protein structure, as manifested by the ring flip motions of aromatic rings. The significance of transient cavities, reflecting an intrinsic dynamic nature within the native state, has also been discussed.

The structure and conformational switching of Rap1B.

Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues.

Synthesis of double-fluorescent labeled prion protein for FRET analysis.

An abnormal form of prion protein (PrP) is considered to be the pathogen in prion diseases. However, the structural details of this abnormal form are not known. To characterize the non-native structure of PrP, we synthesized position-specific double-fluorescent labeled PrP for a fluorescence resonance energy transfer (FRET) experiment. Using FRET, we observed a conformational change in the labeled PrP associated with amyloid fibril formation. The FRET analysis indicated that the distance between fluorescent labeled N- and C-terminal sites of PrP increased upon the formation of amyloid fibrils compared with that of the native state. This approach using FRET analysis is useful for elucidating the structure of abnormal PrP.

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.

Nearly reversible conformational change of amyloid fibrils as revealed by pH-jump experiments.

pH-jump induced conformational transitions between substates of preformed amyloid fibrils made by a fragmented peptide of helix 2 (H2 peptide) of MoPrP were detected, and their kinetics were analyzed using a novel pH-jump apparatus specially designed for observing amyloids. Previously, we reported that H2 peptide formed ordered fibrils with a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils), but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5 (named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed, the CD spectrum changed instantly, but the finally observed ellipticities were clearly distinct from those of pH 7.5 fibrils. Thus, the finally observed state is termed 'pH 7.5-like fibrils'. However, pH 7.5-like fibrils reverted to the conformation very similar to that of the pH 2.9 fibrils when the pH of the solution was restored to 2.9. Then, we examined the kinetics of the nearly reversible conformational changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence stopped-flow, and we observed relatively fast phases (0.7-18 s(-1)). In contrast, the conversion between pH 7.5-like fibrils and pH 7.5 fibrils never occurred (<0.2 day(-1)). Thus, H2 fibrils can be switched readily between distinct conformations separated by a low energy barrier, while a large energy barrier clearly separated the different conformations. These conformational varieties of amyloid fibrils may explain the physical basis of the diversity in prion.

Structure-based discovery of anti-influenza virus A compounds among medicines.

BACKGROUND: Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme. METHODS: We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay. RESULTS: We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase. CONCLUSIONS: Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase. GENERAL SIGNIFICANCE: Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.

Exploring the folding energy landscape with pressure.

The unique role of pressure in protein folding studies is emphasized. Variable-pressure NMR experiments carried out under equilibrium conditions give unique opportunities to explore the energy landscape for protein folding. Intermediate conformers that may appear transiently in the kinetic folding experiments may be stably trapped under pressure, allowing examination of their conformations in site-specific detail with modern NMR spectroscopy. The intimate relationship between the kinetic folding experiment and the equilibrium pressure experiment is described with examples from ubiquitin and hen lysozyme.

A theoretical study of the two binding modes between lysozyme and tri-NAG with an explicit solvent model based on the fragment molecular orbital method.

To examine the stabilities and binding characteristics, fragment molecular orbital (FMO) calculations were performed for the two binding modes of hen egg-white lysozyme with tri-N-acetyl-D-glucosamine (tri-NAG). Solvent effects were considered using an explicit solvent model. For comparison with the computational results, we experimentally determined the enthalpic contribution of the binding free-energy. Our calculations showed that the binding mode observed by X-ray analysis was more stable than the other binding mode by -6.2 kcal mol(-1), where it was found that the interaction of protein with solvent molecules was crucial for this stability. The amplitude of this energy difference was of the same order as the experimental enthalpic contribution. Our detailed analysis using the energies divided into each residue was also consistent with a previous mutant study. In addition, the electron density analysis showed that the formal charge of the lysozyme (+8.0 e) was reduced to +5.16 e by charge transfer with solvent molecules.