Masafumi Hidaka

γ-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional co-factors: nicastrin, Aph-1, and Pen2. It cleaves the transmembrane domains of type-I transmembrane proteins, such as the amyloid precursor protein (APP) and Notch. The cleavage of APP generates the amyloid β peptides (Aβ), which accumulate in patients with Alzheimer's disease. Despite significant research, the exact mechanism of this unique proteolysis, which occurs within the lipid bilayers, is still not fully understood. To study the enzymatic properties of γ-secretase, we have established a yeast reporter system using artificial γ-secretase substrates containing APP or Notch fragments fused to the transcriptional activator Gal4. The γ-secretase activity was evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane-bound substrates, as assessed by the growth of yeast or β-galactosidase activity. Furthermore, we have developed an in vitro assay to identify the different forms of Aβ produced from yeast microsomes. These yeast models provide a platform to screen mutations, genes, and compounds that affect γ-secretase function. By studying the loss of function properties of PS1 familial Alzheimer's disease (FAD) mutants, it is possible to screen for FAD suppressor mutations and γ-secretase modulators (GSMs), in addition to γ-secretase inhibitors (GSIs). In this report, we describe the genetic and biochemical methods used to study γ-secretase activity in the yeast system with the essential steps of the protocol for the video.

γ-Secretase is a protease complex embedded in the cell membrane, consisting of the catalytic presenilin subunits (PS1 or PS2) and three additional co-factors: nicastrin, Aph-1, and Pen2. It cleaves the transmembrane domains of type-I transmembrane proteins, such as the amyloid precursor protein (APP) and Notch. The cleavage of APP generates the amyloid β peptides (Aβ), which accumulate in patients with Alzheimer's disease. Despite significant research, the exact mechanism of this unique proteolysis, which occurs within the lipid bilayers, is still not fully understood. To study the enzymatic properties of γ-secretase, we have established a yeast reporter system using artificial γ-secretase substrates containing APP or Notch fragments fused to the transcriptional activator Gal4. The γ-secretase activity was evaluated by transcriptional activation of reporter genes upon Gal4 release from the membrane-bound substrates, as assessed by the growth of yeast or β-galactosidase activity. Furthermore, we have developed an in vitro assay to identify the different forms of Aβ produced from yeast microsomes. These yeast models provide a platform to screen mutations, genes, and compounds that affect γ-secretase function. By studying the loss of function properties of PS1 familial Alzheimer's disease (FAD) mutants, it is possible to screen for FAD suppressor mutations and γ-secretase modulators (GSMs), in addition to γ-secretase inhibitors (GSIs). In this report, we describe the genetic and biochemical methods used to study γ-secretase activity in the yeast system with the essential steps of the protocol for the video.

Wood ash, a byproduct of woody biomass power generation, has potential as an alternative K fertilizer due to its high K content and pH-raising properties. However, concerns remain about heavy metal contaminants like Cr and the limited understanding of element dynamics in soil–solution–crop systems after wood ash’s application. This study examined the effects of 1% (w/w) wood ash on element dynamics and komatsuna (Brassica rapa var. perviridis) uptake in low-K soil through a pot experiment. XRD was used to analyze mineral composition, SEM-EDS to observe surface and elemental properties, and XANES to examine Cr speciation in wood ash. Soil solution analysis covered macro- and micronutrients, heavy metals, anions, pH, and DOC, while crop element concentrations and aboveground dry weight were also quantified. The chemical speciation of Cu and Cr in a soil solution was modeled using Visual MINTEQ. Wood ash significantly increased K concentrations (from 17 mg/L to 650 mg/L) in the soil solution, along with Ca, Mg, P, and Mo, while reducing Ni, Mn, Zn, and Cd levels. Komatsuna K uptake surged from 123 mg/kg to 559 mg/kg, leading to a 3.31-fold biomass increase. Notably, the Cd concentration in the crops dropped significantly from 0.709 to 0.057 mg/kg, well below the Codex standard of 0.2 mg/kg. Although Cu and Cr concentrations rose in the soil solution, crop uptake remained low due to >99% complexation with fulvic acid, as confirmed by Visual MINTEQ modeling. This study confirms that wood ash is an effective K fertilizer, but emphasizes the need for risk mitigation strategies to ensure safe and sustainable agricultural application.

The emergence of coronavirus disease 2019 (COVID-19) posed a major challenge to healthcare systems worldwide, especially as mutations in the culprit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) complicated the development of vaccines and antiviral drugs. Therefore, the search for natural products with broad anti-SARS-CoV-2 capabilities is an important option for the prevention and treatment of similar infectious diseases. Lectins, which are widely recognized as antiviral agents, could contribute to the development of anti-SARS-CoV-2 drugs. This study evaluated the binding affinity of six lectins (including the cyanobacterial lectin from Microcystis viridis NIES-102 (MVL), and Jacalin, a lectin from the breadfruit, Artocarpus altilis) to the receptor binding domain (RBD) of the spike protein on the original (wild) SARS-CoV-2 and three of its mutants: Alpha, Delta, and Omicron. MVL and Jacalin showed distinct binding affinity to the RBDs of the four SARS-CoV-2 strains. The remaining four lectins (DB1, ConA, PHA-M and CSL3) showed no such binding affinity. Although the glycan specificities of MVL and Jacalin were different, they showed the same affinity for the spike protein RBDs of the four SARS-CoV-2 strains, in the order of effectiveness Alpha > Delta > original > Omicron. The verification of glycan-specific inhibition revealed that both lectins bind to RBDs by glycan-specific recognition, but, in addition, MVL binds to RBDs through protein–protein interactions.

The emergence of the coronavirus 19 (COVID-19) has posed a major challenge to healthcare systems worldwide, especially as COVID-19 mutations complicate the development of vaccines and antiviral drugs. Therefore, the search for natural products with broad anti-COVID-19 capabilities is an important option for the prevention and treatment of infectious diseases. Lectins, which are widely recognized as antiviral agents, could contribute to the development of anti-COVID-19 drugs. In this study, we evaluated the binding ability of a total of six lectins, including MVL from Microcystis viridis NIES-102, to the spike protein RBD of the original (wild) COVID-19 and it’s three mutants: alpha, delta, and omicron. As a result, two lectins, MVL from Microcystis viridis NIES-102 and jacalin from Artocarpus altilis, showed distinct binding ability to the RBDs of the four COVID-19 strains. The other lectins (DB1, ConA, PHA-M and CSL3) did not show any binding ability. Although the glycan specificities of the two lectins, MVL and Jacalin, were different, they showed the same affinity for the RBDs of the four COVID-19 strains, i.e., alpha > delta > original > omicron. The verification of glycan-specific inhibition revealed that jacalin binds to RBDs by glycan-specific recognition, whereas MVL binds to RBDs by protein-protein interactions in addition to glycan-specific recognition.

A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-β peptides (Aβs; Aβ40 and Aβ42), which form fibrils and accumulate in the brain of patients with Alzheimer’s disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aβ secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aβ40 and Aβ42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aβ fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aβ-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.

Amyloid-β peptides (Aβs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer’s disease. Familial Alzheimer’s disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aβs, i.e., Aβ42 and Aβ43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aβ production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aβ production by changing the proportions of Aβ species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.

Although several reports exist on the use of X-ray analysis in vegetables and fruits to examine internal disorders, cavities, and porosity, information on X-ray analysis of qualities, such as texture, is lacking as well as information on X-ray analysis of legumes. Therefore, this study aimed to perform X-ray analysis with sensory and rheometer tests in cooked vegetable soybean (edamame). Edamame is popular worldwide due to its deliciousness and nutritional value. Vascular structures and cracks around them were clearly visualized using X-ray phase-contrast computed tomography (CT) imaging. In addition, we observed the fine structure of the seed coat, which could be important for seed development, germination, and processing. The density in the edamame beans declined as the boiling time increased, promoting a reduction in hardness described in sensory and rheometer tests. The reduction in density proceeded from the gap between cotyledons, the opposite side of the hypocotyl, and the crack. Collectively, the findings show that the high-resolution X-ray phase-contrast CT imaging conducted in a nondestructive manner may help in effectively evaluating the quality of vegetables and in observing the internal structures related to plant development.

Amyloid beta peptides (Aβs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aβ42/43 species is enhanced by familial Alzheimer’s disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aβ production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aβs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.