Biology subjects

Hanazono, Y.

Publications and source records attributed to Hanazono, Y..

4 recordsLinked to original sources

bioRxiv2025

Ataxia-Telangiectasia Mutated Inhibition Enhances Adeno-Associated Virus-Mediated Knock-in Efficiency in Pig Zygotes

A persistent global shortage of transplantable organs has renewed interest in pigs as donors for xenotransplantation and hosts for interspecies organogenesis considering their physiology closely resembles that of humans. Such applications require pigs harboring genetic modifications, including knockouts, human transgenes, and conditional alleles. Although such complex allele combinations can be assembled by sequential editing of fetal fibroblasts, followed by somatic cell nuclear transfer (SCNT), SCNT is often associated with developmental abnormalities. Direct genome editing of pig zygotes offers an alternative route; however, achieving long-fragment knock-in, particularly at multiple loci, remains challenging. Here, adeno-associated virus (AAV) donor vectors for long-fragment knock-in in pig zygotes were evaluated, and tested whether transient inhibition of ataxia-telangiectasia mutated (ATM), which becomes overactivated during AAV transduction, could enhance knock-in efficiency. AAV6 donors supported efficient ACTB-mEGFP knock-in (33%), whereas short-term treatment with the ATM inhibitor AZ32 increased the efficiency to 66%. In addition, AZ32 enhanced simultaneous double knock-in at ACTB and UBB, generating a higher proportion of double-positive blastocysts (52% vs. 19% in controls), without affecting blastocyst formation rates or total cell numbers. Embryo transfer yielded ACTB-mEGFP and UBB-mScarlet-I3 double knock-in fetuses (33%), including an F0 fetus composed almost entirely of double knock-in cells. These results demonstrate that combining AAV donors with transient ATM inhibition is an efficient strategy for long-fragment knock-in at multiple loci in pigs.

bioengineering↗

bioRxiv2025

Transient ATM inhibition enhances knock-in efficiency in hematopoietic stem cells by attenuating the DNA damage response

Precise genome editing in hematopoietic stem cells (HSCs) offers great potential for treating inherited blood disorders, but low knock-in (KI) efficiency, due to HSC quiescence and a preference for non-homologous end joining (NHEJ) and DNA damage-induced apoptosis, remains a major barrier. Here, we demonstrate that transient inhibition of Ataxia-Telangiectasia Mutated (ATM) kinase markedly enhances KI efficiency in mouse HSCs genome-edited with Cas9/RNP and AAV donor DNA. Phosphoproteomic analysis and capillary western blotting revealed that ATM inhibition suppressed the Cas9-AAV-induced ATM activation and subsequent DNA damage response, reduced p53-dependent apoptosis and preserved knock-in competent cells. In transplantation experiments, ATM inhibition preserved long-term engrafting genome-edited HSCs, increasing their frequency from [~]0.3% to [~]40% in secondary recipients - a >100-fold enhancement compared to untreated cells. Furthermore, in an X-SCID mouse model, ATM inhibition enhanced KI efficiency and restored expression of IL-2 receptor {gamma} chain (CD132). These strikingly novel findings highlight transient ATM inhibition as a powerful and clinically relevant approach to enhance KI-mediated genome editing in HSCs, while preserving their long-term repopulating capacity. Key pointsO_LIATM inhibition enhances knock-in efficiency in mouse hematopoietic stem cells C_LIO_LIATM inhibition suppresses Cas9-AAV-induced overactivation of ATM and subsequent p53-dependent apoptosis. C_LI

cell biology↗

bioRxiv2025

Intrinsically Dominant Conformational Diversity in PDZ1 within the Tandem PDZ1-PDZ2 of Human Syntenin-1 Underlined by Crystal Structures.

The intrinsic dynamic asymmetry between homologous PDZ domains in multidomain scaffold proteins offers critical insights into evolutionary mechanisms enabling multivalent partner recognition. Through systematic X-ray crystallographic analysis of human syntenin-1s PDZ1-PDZ2 tandem, we resolve nine high-resolution structures that uncover fundamental differences in conformational plasticity between these sequentially similar domains. Pairwise root-mean-squared deviation (RMSD) analysis of 20 PDZ1 structures across multiple crystal forms reveals substantial structural variability concentrated in the Lys119-Ile125 and Ala181-Glu184 loops - key regions governing ligand specificity within PDZ1s binding cleft. In stark contrast, PDZ2 maintains remarkable structural conservation across all crystallographic environments, indicating divergent evolutionary constraints on these tandem domains. Crucially, comparative analysis of isotropic B-factors demonstrates their inadequacy in capturing the full scope of conformational heterogeneity, emphasizing the necessity of multi-structure comparisons for mapping dynamic landscapes. Molecular dynamics (MD) simulations implemented through GROMACS corroborate these crystallographic observations, showing elevated residue-specific fluctuation (RMSF) values in PDZ1s ligand-binding interface compared to analogous PDZ2 regions. This consistency across experimental and computational approaches confirms that PDZ1s conformational diversity represents an inherent biophysical property rather than crystallographic artifact. The observed dynamic asymmetry suggests a functional division of labor: PDZ1s structural plasticity enables broad ligand recognition via conformational selection mechanisms, while PDZ2s rigid architecture likely stabilizes the tandem domain arrangement. These findings provide an atomic-level rationale for syntenin-1s pleiotropic roles in cellular signaling and establish a structural blueprint for developing domain-selective therapeutics. Given syntenin-1s clinical relevance in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders, our work advances strategies for selectively modulating PDZ1-mediated interactions while preserving PDZ2s scaffolding functions through structure-guided inhibitor design. Highlights{lozenge} Crystal structures of human Syntenin-1s PDZ1-PDZ2 tandem reveal intrinsic conformational plasticity in PDZ1, particularly in ligand-binding loops, contrasting with PDZ2s rigid architecture {lozenge}Pairwise RMSD analysis of 20 PDZ1 structures demonstrates substantial structural variability in the Lys119-Ile125 and Ala181-Glu184 loops, key regions governing ligand specificity {lozenge}Molecular dynamics simulations confirm that PDZ1s conformational diversity is an inherent biophysical property, not a crystallographic artifact {lozenge}The asymmetric dynamics between PDZ1 and PDZ2 suggest a functional division: PDZ1s plasticity enables broad ligand recognition while PDZ2 stabilizes the tandem arrangement {lozenge}These findings provide a structural basis for developing domain-selective Syntenin-1 inhibitors with potential applications in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders

biophysics↗

bioRxiv2023

Elevator motion of chloride ion transporter SLC26A9 induced by STAS domain

The anion exchanger SLC26A9, consisting of the transmembrane (TM) domain and the cytoplasmic STAS domain, plays an essential role in regulating chloride transport across cell membranes. Recent studies have indicated that C-terminal helices block the entrance of the putative ion transport pathway. However, the precise functions of the STAS domain and C-terminal helix, as well as the underlying molecular mechanisms governing the transport process, remain poorly understood. In this study, we performed molecular dynamics simulations of three distinct models of human SLC26A9: full-length (FL), STAS domain removal ({Delta}STAS), and C-terminus removal ({Delta}C), to investigate their conformational dynamics and ion binding properties. Stable binding of ions to the binding sites was exclusively observed in the {Delta}C model in these simulations. Comparing the FL and {Delta}C simulations, the {Delta}C model displayed enhanced motion of the STAS domain. Furthermore, comparing the {Delta}STAS and {Delta}C simulations, the {Delta}STAS simulation failed to exhibit stable ion bindings to the sites despite the absence of the C-terminus blocking the ion transmission pathway in both systems. These results suggest that the removal of the C-terminus not only unblocks the access of ions to the permeation pathway but also triggers STAS domain motion, gating the TM domain to promote ions entry into their binding site. Further analysis revealed that the asymmetric motion of STAS domain leads to the expansion of the ion permeation pathway within the TM domain, resulting in the stiffening of the flexible TM12 helix near the ion binding site. This structural change in the TM12 helix stabilizes chloride ion binding, which is essential for SLC26A9 elevator motion. Overall, our study provides new insights into the molecular mechanisms of SLC26A9 transport and may pave the way for the development of novel treatments for diseases associated with dysregulated ion transport. SIGNIFICANCEWe explored the mechanism by which the human protein SLC26A9 transports chloride in the cell. SLC26A9 is a potential therapeutic target for patients with cystic fibrosis, as by targeting drugs to it, it may be possible to restore chloride ion transport in epithelial cells. To design therapeutic drugs, it is essential to understand how the protein works. Our findings support an elevator-type mechanism, in which chloride ions bind to SLC26A9 inside the cell and are then released by the protein to the extracellular environment. We find that the STAS domain of SLC26A9 has critical roles in binding chloride and induces conformational changes in the transmembrane domain that facilitate chloride transport.

biophysics↗