Yuji O Kamatari

Gifu University | Gidai · Life Science Research Center

We previously reported that N,N-dimethylaniline derivatives are potent ferroptosis inhibitors. Among them, the novel aminoindan derivative GIF-2197-r (the racemate of GIF-2115 (R-form) and GIF-2196 (S-form)) is effective at a concentration of 0.01 μM due to its localization to lysosomes and ferrous ion coordination capacity. The current study demon...

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To as...

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this...

Tannins are involved in the taste of foods and multi bioactivity of traditional herbal medicines. The characteristics of tannins are believed to derive from their connectivity with proteins. However, the mode of interaction between proteins and tannins is not yet understood because of the complexity of the tannin structure. Then this study aimed to...

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via...

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 E40...

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 extracellul...

The antibody G2 specifically binds to four peptides with different amino acid sequences: Pep18mer, Pep8, Pep395, and PepH4P6. To elucidate the multi-specificity of G2, we generated a G2 single-chain Fv (scFv) antibody and analyzed its binding thermodynamics and kinetics to antigen peptides. Our results clearly showed that the recognition of PepH4P6...

Enzootic bovine leukosis (EBL) is a disease caused by bovine leukemia virus (BLV); only a small percentage of BLV-infected cattle develop EBL and present with B-cell lymphosarcoma. There is no vaccine against BLV, treatment for EBL, or method for predicting the possibility of EBL onset, thus making EBL control difficult. Herein, to explore biomarke...

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 de...

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), w...

Introduction Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. Material and Methods The gene encoding the major envelope protein of SPPV was cloned into t...

Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the crystal structure of hen egg lysozyme refolded after denaturation at acidic pH (rHEL) and found a structure different from native HEL (nHEL). The different local conformations of...

Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on...

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for n...

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 generatin...

Propolis, a compound produced by honeybees, has long been used in food and beverages to improve health and prevent diseases. We previously reported that the ethanol extracts of Brazilian green propolis and its constituents artepillin C, kaempferide, and kaempferol mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. Here, we inves...

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 th...

Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexpl...

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate...

Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those fro...

Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conver...

Intermolecular interaction between hPrP and αS was investigated using high-speed atomic force microscopy, dynamic light scattering, and nuclear magnetic resonance. We found that hPrP spontaneously gathered and naturally formed oligomers. Upon addition of monomer αS with a disordered conformation, poly-dispersive property of hPrP was lost, and heter...

Milk extracellular vesicles (EVs) are nanoparticles that contain proteins, mRNAs, microRNAs, DNAs, and lipids that involved in several biological functions. Milk EVs provide proteins that could represent relevant novel biomarkers for monitoring of different diseases such as breast cancer and mastitis in humans and animals, respectively. Bovine leuk...

Nucleic acid aptamers are innovative and promising candidates to block the hallmark event in the prion diseases, that is the conversion of prion protein (PrP) into an abnormal form; however, they need chemical modifications for effective therapeutic activity. This communication reports on the development and biophysical characterization of a small...

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease associated with aggregation of superoxide dismutase 1 (SOD1) protein. More than 160 mutations in human SOD1 have been identified in familial ALS and extensively characterized in previous studies. Here, we investigated the effects of T18S and E40K mutations on p...

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 specifi...

Milk extracellular vesicles (EVs) form an excellent source of mRNAs, microRNAs (miRNAs), proteins, and lipids that represent the physiological and pathological status of the host. Recent studies have reported milk EVs as novel biomarkers for many infectious diseases in both humans and animals. For example, miRNAs in milk EVs from cattle were used f...

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently...

Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrPC) to a pathological (PrPSc) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrPC molecule, inhibiting PrPC-to-PrPSc conversion. Here, we developed a new...

In prion diseases, the causative agent is thought to be solely composed of misfolded prion proteins (PrPSc). It has been shown that PrPSc can convert host-encoded normal isoform of prion protein (PrPC), but the spontaneous emergence of PrPSc is still enigmatic. Certain amyloidogenic proteins have recently been shown to undergo liquid phase separati...

The minimum functional unit of c-Myb for specific DNA binding, c-Myb R2R3, fluctuates largely in solution which is critical for its DNA-binding function. The thermal stability of R2R3 increases with increasing NaCl concentrations and upon DNA binding as well. The DNA-binding affinity of R2R3 is stringently dependent on NaCl concentration, and decre...

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),...

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that lack therapeutic solutions. Here, we show that the molecular chaperone (N,N′-([cyclohexylmethylene]di-4,1-phenylene)bis(2-[1-pyrrolidinyl]acetamide)), designed via docking simulations, molecular dynamics simulations and quantum chemical calculations, slows do...

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for huma...

Significance On the basis of the amino acid sequence, a polypeptide folds into a unique structure. In contrast, aggregation-prone proteins often misfold into distinct aggregate conformations. Interestingly, each distinct aggregate conformation can dictate different phenotypic consequences, such as cellular toxicity. However, the underlying mechanis...

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Intere...

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...

Inhibitors of AKR1B10 belonging to the aldo-keto re-ductase (AKR) superfamily are considered promising candidates for anti-cancer drugs. AKR1B1, a structurally similar isoform of AKR1B10, is involved in glucose metabolism. Thus, selective inhibition of AKR1B10 is required for the development of anti-cancer drugs. In this study, we first compared co...

Supporting Information

Supporting Information

Supporting Information

Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non-native state. The former ligands are termed "stabilizer chaperones" and the latter ones "destabilizer chaperones." Because the stabilization effects are ess...

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...

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,...

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 structur...

We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with residues 174–247 of the chicken prion protein (ChPrPC). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrPC, the original antigenic protein. We immunoscree...

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 minim...

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prio...

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 determin...

A variety of anti-prion compounds have been reported that are effective in ex vivo and in vivo treatment experiments. However, the molecular mechanisms for most of these compounds remain unknown. Here we classified anti-prion mechanisms into four categories: I, specific conformational stabilization; II, non-specific stabilization; III, aggregation;...

The unique role of pressure in protein folding studies is emphasized. The variable-pressure NMR experiments carried out under equilibrium conditions gives 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 pressu...

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...

A new type of flavonoid was isolated from an adzuki bean-water extract by various chromatographic techniques. The chemical structure was determined by ultraviolet-visible (UV-Vis) spectroscopy, mass spectrometry (MS), and various nuclear magnetic resonance (NMR) experiments, and it was found to be unique in that the xanthylium skeleton was present...

Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor pro...

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...

We have used low temperatures (down to -20°C) and high pressures (up to 2000 bar) to populate low-lying excited state conformers of hen lysozyme, and have analyzed their structures site-specifically using (15)N/(1)H two-dimensional HSQC NMR spectroscopy. The resonances of a number of residues were found to be selectively broadened, as the temperatu...

Transmissible spongiform encephalopathies are associated with the conformational conversion of the prion protein from the cellular form (PrP(C)) to the scrapie form. This process could be disrupted by stabilizing the PrP(C) conformation, using a specific ligand identified as a chemical chaperone. To discover such compounds, we employed an in silico...

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and...

A continuous-wave probed laser-induced temperature jump system was constructed and applied to monitor the changes in tryptophan fluorescence of the beta-lactoglobulin during its folding; the kinetic phases were traced from 300 ns to 10 ms after a temperature jump. Notably, an early phase with typical squeezed-exponential characteristics, [exp[-(kt)...

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrPC) to the scrapie form (PrPSc) is still unknown. Here we discovered a chemical chaperone to stabilize the PrPC conformation and identified the hot spots to stop the pathogenic conversion. We co...

The solution structure of the GB1 domain of protein G at a pressure of 2 kbar is presented. The structure was calculated as a change from an energy-minimised low-pressure structure using (1)H chemical shifts. Two separate changes can be characterised: a compression/distortion, which is linear with pressure; and a stabilisation of an alternative fol...

Aggregation of proteins is a serious problem, affecting both human health and industrial production of proteins. We have recently succeeded in showing that association and dissociation of amyloid protofibrils can be controlled reversibly with pressure and the protofibril formation is a slow process toward thermodynamic equilibrium. Extensive studie...

The dissociation and reassociation processes of amyloid protofibrils initiated by pressure-jump have been monitored with real-time (1)H NMR spectroscopy using an intrinsically denatured disulfide-deficient variant of hen lysozyme. Upon pressure-jump up to 2 kbar, the matured protofibrils grown over several months become fully dissociated into monom...

Extensive structural studies using high-pressure NMR spectroscopy have recently been carried out on proteins, which potentially contribute to our understanding of the mechanisms of protein folding. Pressure shifts the conformational equilibrium from higher to lower volume conformers. If the pressure is varied, starting from the folded native struct...

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H...

Although the mechanism of the conformational conversion from the cellular (PrP(C)) to the scrapie (PrP(Sc)) form of animal prion proteins has yet to be elucidated, evidence is accumulating that may provide insight into the conversion process at atomic resolution. Here we show critical aspects of the slow fluctuation dynamics of the recombinant hams...

Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkali...

High-pressure 15N/1H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 m urea, over the pressure range 30–2000 bar. Most 1H and 15N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 ± 0.1...