Biology subjects

Hino, T.

Publications and source records attributed to Hino, T..

8 recordsLinked to original sources

bioRxiv2026

Initiating mammalian reproduction from a single male genome

Sex in mammals is unidirectionally determined by the configuration of sex chromosomes. In contrast, several species, including fishes, can switch sexes in response to environmental cues, illustrating the reproductive advantages of sexual plasticity. Thus, technologies that can bypass the unidirectional nature of mammalian sex determination could open new avenues in reproductive biology. Here, by establishing a method that enables robust Y-chromosome elimination in embryos, we provide a strategy to regulate sex in mice. We show that this approach efficiently induces male-to-female sex reversal by releasing the Y chromosome as micronuclei during early development. When integrated with optimized somatic cell nuclear transfer, it enabled the parallel production of both male and sex-reversed female mice from a single male genome source. Offspring were successfully obtained from crosses between these male and sex-reversed female clones, demonstrating that this "dual-sex cloning" strategy can initiate sexual reproduction solely from a male somatic genome. These findings highlight dual-sex cloning as a valuable platform for securing mammalian reproduction and biodiversity from limited genetic resources.

developmental biology↗

bioRxiv2026

UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7

The AAA-ATPase p97 and its primary adaptor UFD1-NPL4 (UN) unfold ubiquitinated substrates for their proteasomal degradation. Although cryo-EM has shown that substrate processing begins with unfolding of the initiator ubiquitin and its capture by NPL4, the specific contribution of UFD1 to K48-linked ubiquitin chain engagement has not been fully defined. Here we report a 1.31 [A] crystal structure of the human UFD1-UT3 bound to a K48-linked di-ubiquitin mimic, revealing that UFD1-UT3 engages the unfolded initiator ubiquitin via its Nc subdomain and the proximal ubiquitin via its Nn subdomain. AlphaFold3-guided analyses further demonstrate that accessory adaptors FAF1, FAF2, and UBXD7 enhance p97-UN activity through divergent strategies. FAF1 and FAF2 scaffold UFD1-UT3 near the NPL4-tower, whereas UBXD7 stabilizes transient interactions between UFD1 and the ubiquitin chain. Our results provide a comprehensive molecular model for the initiation of p97-mediated substrate unfolding.

biochemistry↗

bioRxiv2023

Isolation and genomic and physiological characterization of Parageobacillus sp. G301, the isolate capable of both hydrogenogenic and aerobic carbon monoxide oxidation

Prokaryotes, known as carbon monoxide (CO) oxidizers, use CO as the carbon or energy source with CO dehydrogenases (CODHs), which are divided into nickel-containing CODH (Ni-CODH) that are sensitive to O2 and molybdenum-containing CODH (Mo-CODH) that are capable of aerobic functioning. The oxygen conditions for CO oxidizers to oxidize CO may be limited because CO oxidizers isolated and characterized so far have either Ni- or Mo-CODH. Here, we report a novel CO oxidizer capable of CO oxidation with both types of CODH based on genomic and physiological characterization of the isolate Parageobacillus sp. G301. This thermophilic facultative anaerobic Bacillota bacterium was isolated from the sediment of a freshwater lake. Genomic analyses showed that G301 was the only isolate possessing both Ni-CODH and Mo-CODH. Genome-based reconstruction of the respiratory machinery and physiological investigation indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), and CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments even without terminal electron acceptors other than protons. As comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus, CO oxidation genes would be retained exclusively for CO metabolism and related respiration. ImportanceMicrobial CO oxidation has received a lot of attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Microbial CO oxidizers have a punctate phylogenetic distribution throughout bacteria and archaea, even in genus-level monophyletic groups. In this study, we demonstrated that the new isolate Parageobacillus sp. G301 is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which had not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, would accelerate research into such CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are optional but not essential genetic elements in the genus Parageobacillus, providing insight into a factor that shapes the mosaic phylogenetic distribution of CO oxidizers, even in genus-level monophyletic groups.

microbiology↗

bioRxiv2022

Mapping Protein Dynamics at High-Resolution with Temperature-Jump X-ray Crystallography

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

biophysics↗

bioRxiv2021

Trim41 is essential for preventing X chromosome chaotic synapsis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential chromosome events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 KO spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome, showing extensive self-synapsis of X chromosome and non-homologous synapsis between the X chromosome and autosomes. Furthermore, the mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 ({Delta}RING-TRIM41) and found that {Delta}RING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result showed that TRIM41 exerts the function on the chromosome axes. In summary, our study revealed that Trim41 is essential for preventing SYCP3 overloading and chaotic synapsis of the X chromosome, suggesting a TRIM41-mediated mechanism for regulating unsyapsed axes during male meiotic progression. Summary statementTrim41-disruption caused abnormal synapsis configuration of the X chromosome and complete infertility in male mice. Thus, TRIM41 prevents the sex chromosome from chaotic synapsis.

developmental biology↗

bioRxiv2021

Production of offspring from azoospermic mice with meiotic failure: Precise biparental meiosis within halved oocytes

While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.

developmental biology↗

bioRxiv2021

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Molecular chirality, discovered by Louis Pasteur in the middle of the 19th century1, is found in most primary and secondary metabolites. Particularly, the so-called natural products are rich in chiral centres2. The stereochemistry of natural products is strictly recognized in living organisms, and is thus closely related to their biological functions. The Diels-Alder (DA) reaction, which forms a six-membered ring with up to four chiral centres, is a fundamental practical reaction for C-C bond formation in synthetic chemistry3. Nature has also adopted this reaction to elaborate the complex structures of natural products using enzymes derived from various progenitor proteins4-7. Although enzymes catalysing the DA reaction, Diels-Alderases (DAases), have attracted increasing attention, little is known about the molecular mechanism by which they control the stereochemistry and perform catalysis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyse intramolecular DA reactions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin8,9. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided dynamic binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, explaining the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyse the stereoselective DA reaction. This study highlights the distinct molecular mechanisms of the enzymatic DA reaction and its stereoselectivity experimentally and computationally. We anticipate that clarified molecular mechanism herein provides not only the basic understanding how these important enzymes work but also the guiding principle to create artificial enzymes that produce designer bioactive molecules.

biochemistry↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗