The way forward for neuroethics in Japan: A review of five topics surrounding present challenges.

Neuroethics is the study of how neuroscience impacts humans and society. About 15 years have passed since neuroethics was introduced to Japan, yet the field of neuroethics still seeks developed methodologies and an established academic identity. In light of progress in neuroscience and neurotechnology, the challenges for Japanese neuroethics in the 2020 s can be categorized into five topics.

Asteltoxin inhibits extracellular vesicle production through AMPK/mTOR-mediated activation of lysosome function.

Cancer cells secrete aberrantly large amounts of extracellular vesicles (EVs) including exosomes, which originate from multivesicular bodies (MVBs). Because EVs potentially contribute to tumor progression, EV inhibitors are of interest as novel therapeutics. We screened a fungal natural product library.

MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.

Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs, often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur.

Crystal structure of a GH1 β-glucosidase from Hamamotoa singularis.

A GH1 β-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.

Val143 of human ribonuclease H2 is not critical for, but plays a role in determining catalytic activity and substrate specificity.

Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity.

Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli.

FtsZ, a tubulin-like GTPase, is essential for bacterial cell division. In the presence of GTP, FtsZ polymerizes into filamentous structures, which are key to generating force in cell division. However, the structural basis for the molecular mechanism underlying FtsZ function remains to be elucidated.

Unlike the counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA.

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor.

Two RNase H2 Mutants with Differential rNMP Processing Activity Reveal a Threshold of Ribonucleotide Tolerance for Embryonic Development.

RNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth.

Development of Wearable Sheet-Type Shear Force Sensor and Measurement System that is Insusceptible to Temperature and Pressure.

A sheet-type shear force sensor and a measurement system for the sensor were developed. The sensor has an original structure where a liquid electrolyte is filled in a space composed of two electrode-patterned polymer films and an elastic rubber ring. When a shear force is applied on the surface of the sensor, the two electrode-patterned films mutually move so that the distance between the internal electrodes of the sensor changes, resulting in current increase or decrease between the electrodes.

Formation of the high-affinity calcium binding site in pro-subtilisin E with the insertion sequence IS1 of pro-Tk-subtilisin.

Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability.

Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain.

Tk-subtilisin (Gly70-Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk-subtilisin is produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide.

Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin.

Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin.

Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon.

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (∆IS1-Pro-TKS) and its active-site mutants (∆IS1-Pro-S324A and ∆IS1-Pro-S324C) were constructed and characterized.

Requirement of Ca(2+) ions for the hyperthermostability of Tk-subtilisin from Thermococcus kodakarensis.

Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca(2+) ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin.