Biology subjects

Konno, H.

Publications and source records attributed to Konno, H..

11 recordsLinked to original sources

bioRxiv2026

BioAFMviewer AFM Analysis Arena A3 - an interactive software platform for automated analysis of HS-AFM imaging

High-speed atomic force microscopy (HS-AFM) experiments provide unique insights into nanoscale biological processes by directly visualizing the surface topography dynamics of biomolecules, such as proteins, over extended periods of time and generating large imaging data sets. However, the lack of computational methods for automated data analysis has thus far limited the full utilization of these data for the quantitative understanding of single-molecule dynamic. Here, we present the AFM Analysis Arena (A3) within BioAFMviewer as a software platform for automated, high-throughput quantitative analysis of AFM topographic imaging data within a user-friendly interactive graphical interface, implementing a convenient workflow from raw HS-AFM (AFM) imaging to statistical analysis. In several applications to HS-AFM imaging data, we demonstrate methods of automatized molecule detection and dynamic tracking to highlight the available capacities, aiming to transform HS-AFM measurements from observations towards an analytical tool in nanoscale biology.

biophysics↗

bioRxiv2026

Structure and function of human NXPE1, a sialic acid O-acetyltransferase

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

immunology↗

bioRxiv2025

Mycophenolate mofetil reduces the branching of microglial processes

Microglia, the resident immune cells in the central nervous system, play important roles not only in immune response but also in neurogenesis, synaptogenesis, and neural circuit formation. Microglia also surveil the brain environment via elongation and retraction of their processes. Previously, we found that the purine salvage pathway is involved in the regulation of morphology and dynamics of the microglial cell line BV2. Here, we show that intraperitoneal administration of mycophenolate mofetil (MMF), an inosine monophosphate dehydrogenase (IMPDH) inhibitor, reduces microglial branching during postnatal development. Imaging mass spectrometry analysis revealed that MMF administration decreases guanosine nucleotides in the brain. Interestingly, despite the essential role of guanosine nucleotides in cellular proliferation, MMF administration did not significantly affect microglial proliferation. On the other hand, MMF administration attenuated the level of GTP-bound forms of RhoA and Rac1 small GTPases. Notably, MMF administration decreased the number of branches, while process length remained unaffected. Since microglial branching affects microglial complexity and diversity, our findings suggest that guanosine nucleotide production is essential for generating proper microglial diversity.

neuroscience↗

bioRxiv2025

Flexible Motion of T7 bacteriophage Tail Fibers Suggest a Dynamic Viral Infection Mechanism

Viruses are nanoscale infectious agents capable of specifically targeting and reprograming host cells. A unique group of viruses, bacteriophages, have regained popularity in research partly due to the rising number of multidrug-resistant bacterial infections. Phages could potentially replace antibiotics, but only if we understand every detail of their structure and infection cycle. T7 bacteriophages are a group of dsDNA viruses, which infect E. coli bacteria. T7 virions are comprised of an icosahedral protein shell which encapsulates the genomic DNA, and a tail-fiber complex which is primarily used for target recognition and DNA injection. The virus has six" L"-shaped, [~]40 nm long fibers (gp17 protein trimers) attached to the tail-tube, which are thought to be essential for initial host recognition and possibly surface exploration. Using high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations combined with small angle X-ray scattering (SAXS) we observed the molecular structure and movements of isolated tail fibers. Firstly, we have identified a hinge region within the fibers, which makes them highly flexible, allowing the bending of their distal region. Furthermore, we have observed the dynamic triple helical coiled coil structure of the proximal region, which would allow fiber rotation. These two points of flexibility allow a more efficient and highly dynamic host recognition and virus anchoring process. The observed flexibility might allow host surface exploration by walking. Such flexibility in the host recognition machinery may not be unique to T7 bacteriophages, getting us one step closer to understanding the intricate details of virus-host interactions.

biophysics↗

bioRxiv2025

Flexible fitting to infer atomistic-precision models of large-amplitude conformational dynamics in biomolecules from high-speed atomic force microscopy imaging

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes of the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton size protein filament demonstrate versatility of NMFF-AFM modelling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step towards automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the standalone user-friendly interactive BioAFMviewer software opens the opportunity for broad range applications to facilitate the understanding of resolution-limited HS-AFM measurements.

biophysics↗

bioRxiv2024

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

bioRxiv2024

Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations.

Classical Anfinsen's dogma states that a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, recent advances have revealed that single amino acid sequences can fold into two or more conformations. Here, we propose a novel approach to design a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal {beta}-strand and DHFR lacking the N-terminal {beta}-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analysis, including AFM imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. In addition, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a new direction for creating artificial proteins with on-off behaviors.

biochemistry↗

bioRxiv2022

Structural dynamics of E6AP E3 ligase HECT domain and involvement of flexible hinge loop in ubiquitin chain synthesis mechanism

Ubiquitin (Ub) ligases E3 are an important factor in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe containing the E2-binding site and a C-lobe containing the catalytic Cys residue that forms a thioester bond with Ub. These two lobes are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of this type of E3 ligases remain unclear. Here, we provide the first direct demonstration of structural dynamics of the E6AP HECT domain using high-speed atomic force microscopy. We also investigated structural dynamics of hinge loop flexibility restricted HECT domain, and we found that flexibility of the E6AP hinge loop has a great impact not only on its structural dynamics but also on the formation of free Ub chains mediated by E3-E3 interactions.

biophysics↗

bioRxiv2021

Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo- SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and disrupt specific membranes. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/438527v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@17ef3f4org.highwire.dtl.DTLVardef@1c24d41org.highwire.dtl.DTLVardef@15e071aorg.highwire.dtl.DTLVardef@24740e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones. C_LIO_LICas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes. C_LIO_LIVulnerability of the specific cytoplasmic membranes to the selective Cas toxin that is the most remarkable feature of cytotoxicity of Cas toxin on plant cells. C_LI

biochemistry↗

bioRxiv2020

The SYP123-VAMP727 SNARE complex is involved in the delivery of inner cell wall components to the root hair shank in Arabidopsis

A root hair is a long tubular protrusion from a root hair cell established via tip growth, which is accomplished by the polarized deposition of membranous and cell wall components at the root hair apex accompanied by simultaneous hardening of the shank. The polarized secretion of materials to the root hair apex is well investigated; however, little is known about the deposition of inner cell wall materials at the root hair shank. We have previously reported that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2)/ROP10 signaling is required for the regulation of cortical microtubule construction and the deposition of inner cell wall components at the root hair shank during hardening. To unravel the alternate secretion mechanism for delivery of the inner cell wall components to root hair shank, here, we demonstrate that root hair-specific Qa-SNARE, SYP123, localizes to the subapical zone and shank of elongating root hairs in Arabidopsis. SYP123-mediated root hair elongation was inhibited by the FAB1 inhibitor YM201636, and inhibition of PtdIns(3,5)P2 production impaired the plasma membrane localization of SYP123. We also showed that SYP123 forms a SNARE complex with VAMP727 on the plasma membrane, and syp123 and vamp727 mutants exhibited lower cell wall stiffness in the root hair shank because of impaired deposition of inner cell wall components. These results indicate that SYP123/VAMP727-mediated secretion is involved in the transport of inner cell wall components for hardening of the root hair shank.

plant biology↗

bioRxiv2018

Proteomic Analysis of NRROS Interactome Reveals the Presence of Chaperones and Mediators of the ERAD Pathway

Negative regulator of reactive oxygen species (NRROS, previously called LRRC33) is a leucine-rich repeat (LRR) domain containing, ER-resident transmembrane protein expressed primarily in lymphoid organs, especially in myeloid cells. We have previously demonstrated that NRROS regulates reactive oxygen species production by phagocytic cells by mediating degradation of NOX2 (gp91phox), a component of NOX2 complex responsible for the oxidative burst in these cells. Since LRR is the only functional domain in NRROS, it is likely to interact with other proteins for its biological functions. Here, by performing immunoprecipitation of NRROS and mass spectrometric analysis, we describe the NRROS interactome in macrophages and demonstrate that NRROS interacts with molecular chaperones/co-chaperones and mediators of the endoplasmic reticulum associated degradation (ERAD) pathway such as calnexin, suggesting a broader role for NRROS in protein biosynthesis and the ER quality control machinery.

biochemistry↗