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Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the numerous studies on the inhibition of amyloid formation, the prevention and treatment of a majority of amyloid-related disorders are still challenging. In this study, we investigated the effects of various plant extracts on amyloid formation of α-synuclein. We found that the extracts from Eucalyptus gunnii are able to inhibit amyloid formation, and to disaggregate preformed fibrils, in vitro. The extract itself did not lead cell damage. In the extract, miquelianin, which is a glycosylated form of quercetin and has been detected in the plasma and the brain, was identified and assessed to have a moderate inhibitory activity, compared to the effects of ellagic acid and quercetin, which are strong inhibitors for amyloid formation. The properties of miquelianin provide insights into the mechanisms controlling the assembly of α-synuclein in the brain.
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The probability of successful drug discovery is declining, and research and development costs are increasing. To solve these problems, pharmaceutical companies tend to in-license seeds from venture companies and academia. Therefore, academia's role in drug discovery is extremely important. Tottori University started a "Next-Generation Research Support Project (Strategic Research Support Project)" in 2020, developing a translational research system to promote drug discovery. In this project, we established a research and development infrastructure, such as seed registration, construction of drug research and development support, and research fund allocation. The registered seed were converted into project, and the project implemented this research and development system, and evaluated and verified its results. Twenty-two seeds were converted into projects and portfolios were constructed. Research funds were allocated to eight prioritized projects. Each project raised the research and development stages. From the overall portfolio, one project with the Japan Agency for Medical Research and Development (AMED) Drug Discovery Booster Project, and three projects with Seeds A of the AMED Translational Research Strategic Promotion Program were adopted. Additionally, a new low-molecular weight chaperone drug against GM1-gangliosidosis was out-licensed to an overseas pharmaceutical company. The strength of this system was the strategic allocation of research funds and the accompanying support that leveraged internal and external resources with the PM and researchers at its core. This system achieved certain results in promoting drug discovery; however, resource optimization of specialized personnel needs to be strengthened in the future. In this report, we summarized the efforts of translational research in Japan and around the world. In addition, the translational research efforts of Japanese academia and Tottori University were compared and the current status was summarized.
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Cell-to-cell spreading of misfolded α-synuclein (αSYN) is supposed to play a key role in the pathological progression of Parkinson's disease (PD) and other synucleinopathies. Receptor-mediated endocytosis has been shown to contributes to the uptake of αSYN in both neuronal and glial cells. To determine the receptor involved in αSYN endocytosis on the cell surface, we performed unbiased, and comprehensive screening using a membrane protein library of the mouse whole brain combined with affinity chromatography and mass spectrometry. The candidate molecules hit in the initial screening were validated by co-immunoprecipitation using cultured cells; sortilin, a vacuolar protein sorting 10 protein family sorting receptor, exhibited the strongest binding to αSYN fibrils. Notably, the intracellular uptake of fibrillar αSYN was slightly but significantly altered, depending on the expression level of sortilin on the cell surface, and time-lapse image analyses revealed the concomitant internalization and endosomal sorting of αSYN fibrils and sortilin. Domain deletion in the extracellular portion of sortilin revealed that the ten conserved cysteines (10CC) segment of sortilin was involved in the binding and endocytosis of fibrillar αSYN; importantly, pretreatment with a 10CC domain-specific antibody significantly hindered αSYN fibril uptake. The presence of sortilin in the core structure of Lewy bodies and glial cytoplasmic inclusions in the brain of synucleinopathy patients was confirmed via immunohistochemistry, and the expression level of sortilin in mesencephalic dopaminergic neurons may be altered with disease progression. These results provide compelling evidence that sortilin acts as an endocytic receptor for pathogenic form of αSYN, and yields important insight for the development of disease-modifying targets for synucleinopathies.
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Proteínas Adaptadoras del Transporte Vesicular , Enfermedad de Parkinson , Sinucleinopatías , Animales , Ratones , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , alfa-Sinucleína/metabolismo , Proteínas Portadoras , Enfermedad de Parkinson/metabolismoRESUMEN
There is considerable interest in drug discovery targeting the aggregation of α-synuclein (αSyn) since this molecular process is closely associated with Parkinson's disease. However, inhibiting αSyn aggregation remains a major challenge because of its highly dynamic nature which makes it difficult to form a stable binding complex with a drug molecule. Here, by exploiting Random non-standard Peptides Integrated Discovery (RaPID) system, we identified a macrocyclic peptide, BD1, that could interact with immobilized αSyn and inhibit the formation of fibrils. Furthermore, improving the solubility of BD1 suppresses the co-aggregation with αSyn fibrils while it kinetically inhibits more effectively without change in their morphology. We also revealed the molecular mechanism of kinetic inhibition, where peptides bind to fibril ends of αSyn, thereby preventing further growth of fibrils. These results suggest that our approach for generating non-standard macrocyclic peptides is a promising approach for developing potential therapeutics against neurodegeneration.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Amiloide/química , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Péptidos/farmacología , CinéticaRESUMEN
Intracellular aggregation of α-synuclein is a major pathological feature of Parkinson's disease. In this study, we show that the polyphenols delphinidin and rosmarinic acid suppress intracellular aggregation of α-synuclein in a mouse neuron cell model when added under oxidative stress conditions. To enhance the detection threshold of this preventive effect of the two polyphenols, we generated a new strain of "aggregation prone model cells" that tended to show prominent α-synuclein aggregation even under normal conditions. Using this new highly sensitive cell line, we demonstrate that addition of delphinidin to model cell cultures effectively suppresses the formation of intracellular α-synuclein aggregates. Flow cytometric analysis shows that adding delphinidin decreases the fraction of "dying cells," cells that were alive but in a damaged state. Our findings suggest the possibility of using polyphenols to prevent and treat the symptoms correlated with the onset of Parkinson's disease. Additionally, our aggregation-prone cell model may be used in future studies to probe numerous neurodegenerative diseases with high sensitivity.
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Enfermedad de Parkinson , alfa-Sinucleína , Ratones , Animales , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Polifenoles/farmacología , Neuronas/metabolismo , Ácido RosmarínicoRESUMEN
The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these "transformed" fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form.
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Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Humanos , Ácidos/metabolismo , Amiloide/química , Amiloide/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Mid-infrared tunable coherent light sources are used in various laser applications, such as trace gas detection, laser processing, and biomedical diagnostics. This study demonstrates mid-infrared generation in the 8.3-11 µm (i.e., 900-1200 cm-1) spectral range by configuring intracavity difference-frequency generation (DFG) using ZnGeP2 (ZGP) in an electronically tuned Cr:ZnSe laser. The broad tunability is achieved with the maximum pulse energies exceeding 100 µJ by combining the electronic wavelength tuning with sligh angle adjustments (Δθ < 0.5°) of ZGP under the spectral noncritical phase-matching condition of the nonlinear material. The proposed DFG method is generalized to give access to a significant fraction of the molecular fingerprint region by utilizing selenide compounds (e.g., AgGaSe2, CdSe) in addition to ZGP, revealing the remarkable potential of ultrabroadband electronic mid-infrared scanning for numerous spectroscopic applications.
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This Letter presents the first, to the best of our knowledge, demonstration of noncritically birefringent-phase-matched parametric downconversion, which is associated with stimulated emission via vibronic transition in a laser gain medium. The so-called self-difference frequency generation is realized along the a-axis of a Cr:CdSe single crystal pumped by a Tm:YAG laser pulse at 2.013 µm, directly producing an infrared spectrum centered at 9 µm with the maximized effective nonlinearity. The light source, which benefits from the broad vibronic spectroscopic properties together with the wide transparency range of the host material, is expected to generate noncritically phase-matched, mid-infrared spectra beyond 20 µm along with birefringence engineering in the solid solution Cr:CdSxSe1-x.
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Aggregation of intrinsically disordered α-synuclein (αSN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of αSN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of αSN mutants under two types of liposomes, 1,2-Dioleoyl-sn-glycero-3-Phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of αSNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of αSNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of αSN, which is rationalized by the amyloidogenic structure of αSN and condensation-dilution of local αSN concentration. Finally, we propose physicochemical properties of αSN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.
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The development of mid-infrared (mid-IR) tunable lasers has been driving various laser spectroscopic technologies. Herein, we report wavelength-scanning cavity ring-down spectroscopy (WS-CRDS) in the mid-IR region using an electronically tuned Cr:ZnSe (ET-Cr:ZnSe) laser, which could achieve a nanosecond pulse operation, with broad wavelength tuning of 2-3 µm. This allowed WS-CRDS-induced trace detection of the refrigerant, CH2F2. A CH2F2 detection limit of 0.66 ppm (3σ), and the detection of trace H2O in CH2F2 was realized using the broad wavelength-tuning range feature, demonstrating the effectiveness of the ET-Cr:ZnSe laser in WS-CRDS. We believe that our method would accelerate the development of various trace-gas detection technologies.
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We previously show that fatty acid-binding protein 3 (FABP3) triggers α-synuclein (Syn) accumulation and induces dopamine neuronal cell death in Parkinson disease mouse model. But the role of fatty acid-binding protein 7 (FABP7) in the brain remains unclear. In this study we investigated whether FABP7 was involved in synucleinopathies. We showed that FABP7 was co-localized and formed a complex with Syn in Syn-transfected U251 human glioblastoma cells, and treatment with arachidonic acid (100 M) significantly promoted FABP7-induced Syn aggregation, which was associated with cell death. We demonstrated that synthetic FABP7 ligand 6 displayed a high affinity against FABP7 with Kd value of 209 nM assessed in 8-anilinonaphthalene-1-sulfonic acid (ANS) assay; ligand 6 improved U251 cell survival via disrupting the FABP7-Syn interaction. We showed that activation of phospholipase A2 (PLA2) by psychosine (10 M) triggered oligomerization of endogenous Syn and FABP7, and induced cell death in both KG-1C human oligodendroglia cells and oligodendrocyte precursor cells (OPCs). FABP7 ligand 6 (1 M) significantly decreased Syn oligomerization and aggregation thereby prevented KG-1C and OPC cell death. This study demonstrates that FABP7 triggers α-synuclein oligomerization through oxidative stress, while FABP7 ligand 6 can inhibit FABP7-induced Syn oligomerization and aggregation, thereby rescuing glial cells and oligodendrocytes from cell death.
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Proteína de Unión a los Ácidos Grasos 7/metabolismo , Neuroglía/metabolismo , Oligodendroglía/metabolismo , Estrés Oxidativo/fisiología , alfa-Sinucleína/metabolismo , Animales , Ácido Araquidónico/farmacología , Muerte Celular/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Fosfolipasas A2/efectos de los fármacos , Unión Proteica/fisiología , Psicosina/farmacologíaRESUMEN
Many plants, including fruits and vegetables, release biogenic gases containing various volatile organic compounds such as ethylene (C2H4), which is a gaseous phytohormone. Non-destructive and in-situ gas sampling technology to detect trace C2H4 released from plants in real time would be attractive for visualising the ageing, ripening, and defence reactions of plants. In this study, we developed a C2H4 detection system with a detection limit of 0.8 ppb (3σ) using laser absorption spectroscopy. The C2H4 detection system consists of a mid-infrared quantum cascade laser oscillated at 10.5 µm, a multi-pass gas cell, a mid-IR photodetector, and a gas sampling system. Using non-destructive and in-situ gas sampling, while maintaining the internal pressure of the multi-pass gas cell at low pressure, the change in trace C2H4 concentration released from apples (Malus domestica Borkh.) can be observed in real time. We succeeded in observing C2H4 concentration changes with a time resolution of 1 s, while changing the atmospheric gas and surface temperature of apples from the 'Fuji' cultivar. This technique allows the visualisation of detailed C2H4 dynamics in plant environmental response, which may be promising for further progress in plant physiology, agriculture, and food science.
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Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol-7âOâglycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α-synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.
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Amiloide , Polifenoles , alfa-Sinucleína , Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Espectroscopía de Resonancia Magnética , Polifenoles/química , Polifenoles/metabolismo , Conformación Proteica , Solubilidad , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
α-synuclein (αSyn) is a protein known to form intracellular aggregates during the manifestation of Parkinson's disease. Previously, it was shown that αSyn aggregation was strongly suppressed in the midbrain region of mice that did not possess the gene encoding the lipid transport protein fatty acid binding protein 3 (FABP3). An interaction between these two proteins was detected in vitro, suggesting that FABP3 may play a role in the aggregation and deposition of αSyn in neurons. To characterize the molecular mechanisms that underlie the interactions between FABP3 and αSyn that modulate the cellular accumulation of the latter, in this report, we used in vitro fluorescence assays combined with fluorescence microscopy, transmission electron microscopy, and quartz crystal microbalance assays to characterize in detail the process and consequences of FABP3-αSyn interaction. We demonstrated that binding of FABP3 to αSyn results in changes in the aggregation mechanism of the latter; specifically, a suppression of fibrillar forms of αSyn and also the production of aggregates with an enhanced cytotoxicity toward mice neuro2A cells. Because this interaction involved the C-terminal sequence region of αSyn, we tested a peptide derived from this region of αSyn (αSynP130-140) as a decoy to prevent the FABP3-αSyn interaction. We observed that the peptide competitively inhibited binding of αSyn to FABP3 in vitro and in cultured cells. We propose that administration of αSynP130-140 might be used to prevent the accumulation of toxic FABP3-αSyn oligomers in cells, thereby preventing the progression of Parkinson's disease.
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Amiloide/antagonistas & inhibidores , Proteína 3 de Unión a Ácidos Grasos/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/farmacología , Agregación Patológica de Proteínas/prevención & control , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Animales , Proteína 3 de Unión a Ácidos Grasos/genética , Humanos , Ratones , Neuroblastoma/genética , Neuroblastoma/metabolismo , Células Tumorales Cultivadas , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/genéticaRESUMEN
Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-µM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.
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Amiloide/biosíntesis , Polifosfatos/farmacología , Agregado de Proteínas/efectos de los fármacos , alfa-Sinucleína/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , alfa-Sinucleína/metabolismoRESUMEN
The thermodynamic hypothesis of protein folding, known as the "Anfinsen's dogma" states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen's dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer's and Parkinson's diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen's intramolecular folding universe and the intermolecular misfolding universe.
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Amiloide/química , Amiloide/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos/fisiología , Amiloidosis/etiología , Amiloidosis/metabolismo , Precipitación Química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Concentración Osmolar , Agregación Patológica de Proteínas/etiología , Agregación Patológica de Proteínas/metabolismo , Conformación Proteica , Multimerización de Proteína/fisiología , Termodinámica , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
α-synuclein accumulation into dopaminergic neurons is a pathological hallmark of Parkinson's disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3) is critical for α-synuclein uptake and propagation to accumulate in dopaminergic neurons. FABP3 is abundant in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D2L). Here, we investigated the importance of dopamine D2L receptors in the uptake of α-synuclein monomers and their fibrils. We employed mesencephalic neurons derived from dopamine D2L -/-, dopamine D2 receptor null (D2 null), FABP3-/-, and wild type C57BL6 mice, and analyzed the uptake ability of fluorescence-conjugated α-synuclein monomers and fibrils. We found that D2L receptors are co-localized with FABP3. Immunocytochemistry revealed that TH+ D2L-/- or D2 null neurons do not take up α-synuclein monomers. The deletion of α-synuclein C-terminus completely abolished the uptake to dopamine neurons. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. D2L and FABP3 were also critical for α-synuclein fibrils uptake. D2L and accumulated α-synuclein fibrils were well co-localized. These data indicate that dopamine D2L with a caveola structure coupled with FABP3 is critical for α-synuclein uptake by dopaminergic neurons, suggesting a novel pathogenic mechanism of synucleinopathies, including Parkinson's disease.
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Cu/Zn superoxide dismutase (SOD1) mutations are associated with amyotrophic lateral sclerosis (ALS). SOD1-positive aggregates in motor neurons, as well as proteins that interact with the aggregates are presumably involved in ALS neurotoxicity. We used a proteomics approach to compare differences in protein expression in spinal cord homogenates from non-transgenic (NTG) and ALS model mice. Using the homogenates, we identified proteins that interacted with SOD1 seeds in vitro. We assessed differences in SOD1-interacting proteins in cell cultures treated with proteasome or autophagy inhibitor. In the first experiment, intermediate filamentous and small heat shock proteins were upregulated in glial cells. We identified 26 protein types that interacted with aggregation cores in ALS model homogenates, and unexpectedly, 40 proteins in were detected in NTG mice. In cell cultures treated with proteasome and autophagy inhibitors, we identified 16 and 11 SOD1-interacting proteins, respectively, and seven proteins in untreated cells. These SOD1-interacting proteins were involved in multiple cellular functions such as protein quality control, cytoskeletal organization, and pathways involved in growth factor signaling and their downstream cascades. The complex interactions between pathways could cause further dysregulation, ultimately leading to fatal cellular dysfunction in ALS.
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Esclerosis Amiotrófica Lateral , Superóxido Dismutasa , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Mutación , Proteolisis , Médula Espinal/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/â2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.
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Neurological dementias such as Alzheimer's disease and Lewy body dementia are thought to be caused in part by the formation and deposition of characteristic insoluble fibrils of polypeptides such as amyloid beta (Aß), Tau, and/or α-synuclein (αSyn). In this context, it is critical to suppress and remove such aggregates in order to prevent and/or delay the progression of dementia in these ailments. In this report, we investigated the effects of spearmint extract (SME) and rosmarinic acid (RA; the major component of SME) on the amyloid fibril formation reactions of αSyn, Aß, and Tau proteins in vitro. SME or RA was added to soluble samples of each protein and the formation of fibrils was monitored by thioflavin T (ThioT) binding assays and transmission electron microscopy (TEM). We also evaluated whether preformed amyloid fibrils could be dissolved by the addition of RA. Our results reveal for the first time that SME and RA both suppress amyloid fibril formation, and that RA could disassemble preformed fibrils of αSyn, Aß, and Tau into non-toxic species. Our results suggest that SME and RA may potentially suppress amyloid fibrils implicated in the progression of Alzheimer's disease and Lewy body dementia in vivo, as well.