Biology subjects

Kawahara, K.

Publications and source records attributed to Kawahara, K..

7 recordsLinked to original sources

bioRxiv2026

Active and unusually expanded PIF/Harbinger transposable elements in the Caenorhabditis inopinata genome

BackgroundTransposable elements (TEs) serve as powerful drivers of genome innovation but also threaten genome integrity. The PIF/Harbinger superfamily is distinctive among DNA transposons because mobilisation typically requires proteins, a DDE transposase and a MADF DNA-binding protein. Caenorhabditis inopinata, the closest known relative of C. elegans, has a TE-rich genome and lacks multiple components of the ERGO-1-class endogenous small-RNA pathway, making it a useful system for examining TE dynamics in a distinct host context. We identified a spontaneous dumpy mutant of C. inopinata caused by insertion of a PIF/Harbinger-family element into the coding region of Cin-dpy-11. The inserted element, designated Harbinger-1M_cIno, belongs to the Turmoil2 lineage originally defined in C. elegans and retains a MADF domain but lacks a recognisable DDE transposase ORF. Genome-wide curation recovered 258 related copies, revealing a strongly asymmetric family structure. Short noncoding derivatives were predominant, MADF-bearing derivatives were expanded and only one DDE-bearing locus retained an apparently intact transposase gene, suggesting that DDE and MADF functions are partitioned across distinct elements and may be supplied in trans during mobilisation. We also identified a second PIF/Harbinger-derived family, Harbinger-2M_cIno, associated with the Turmoil1 lineage. This family comprises 1,376 copies and therefore records substantial past amplification, but it lacks a detectable DDE source, shows greater sequence divergence and more degraded terminal structures than Harbinger-1M_cIno. Together, these data indicate that the two PIF/Harbinger lineages in C. inopinata differ not in whether amplification occurred, but in when it occurred and whether present-day mobilisation competence has been retained.

genetics↗

bioRxiv2026

Molecular basis of collagen triple helix recognition by VWF A-like domain 2 of collagen VII: Implications for interlaced anchoring fibril formation

Anchoring fibrils formed by collagen VII play a critical role in stabilizing the dermal-epidermal junction. The N-terminal non-collagenous (NC1) domain of collagen VII binds firmly to basement membrane components including collagen IV and has also been reported to interact with mesenchymal fibrillar collagens via its von Willebrand factor A-like domain 2 (A2 domain). To elucidate how collagen VII recognizes fibrillar collagen, we performed yeast two-hybrid screening using a triple-helical random peptide library, which resulted in the identification of a Met-Gly-{Phi} ({Phi}; aromatic amino acid residue) motif. Biochemical analysis with synthetic triple-helical peptides revealed a binding preference of Trp > Phe as the {Phi} residue by the A2 domain despite Trp being absent in native collagens. The crystal structure of the A2 domain in complex with the Nle (Met surrogate)-Gly-Trp-containing peptide revealed a unique mechanism by which two distinct hydrophobic pockets of the A2 domain accommodate the Nle and Trp residues corresponding to the Met-Gly-{Phi} motif, engaging all three chains of the triple helix. Subsequent molecular dynamics simulations demonstrated that the A2 domain recognizes the corresponding native Met-Gly-Phe motif in a similar manner, but with lower affinity, implying a transient interaction with mesenchymal collagens. The findings obtained in this work suggest models in which transient A2-triple helix interaction promotes the recruitment of collagen I and III fibrils into the arc-shaped structure of anchoring fibrils. This also provides a foundation for linking structural understanding to skin fragility diseases caused by collagen VII dysfunction.

biochemistry↗

bioRxiv2025

Triple-helical ligands selectively targeting the closed αI domain of integrin α2β1

Integrins 1{beta}1, 2{beta}1, 10{beta}1, and 11{beta}1 are known to recognize the GxxGEx motif of the collagen triple helix through their I domains, with the Glu residue engaging the domain via a divalent metal cation. The binding amino acid sequences containing these motifs, identified from native collagen, exhibit low subtype selectivity. Here we identify novel triple-helical peptides that selectively bind the 2I domain independently of Glu and metal ions. Yeast two-hybrid screening of randomized triple-helical peptide libraries yielded non-natural sequences in which the canonical Glu was replaced by aliphatic residues such as Met. X-ray crystal structural analysis of the representative variant GFOGMR in complex with the 2I domain revealed a new binding mode. In this mode, peptides are recognized by the closed, inactive conformation of the 2I domain without metal coordination. This cryptic interaction, likely unused by native ligands, provides a new basis for the design of subtype-specific ligands targeting collagen-binding integrins.

biochemistry↗

bioRxiv2024

High-resolution Cryo-EM visualises hydrated type I and IV pilus structures from enterotoxigenic Escherichia coli

To efficiently colonise intestinal epithelium, enteric pathogens utilise a wide variety of pilus filaments, including those synthesised by the chaperone-usher or type IV pilus assembly pathway. Despite the importance of these filaments as potential drug and vaccine targets, their large size and dynamic nature present challenges for atomic-resolution structural determination. Here, we used cryo-electron microscopy (cryo-EM) and whole genome sequencing to identify and determine the structures of type I and IV pili expressed in enterotoxigenic Escherichia coli. The well-defined cryo-EM maps at resolutions of 2.20 and 1.78 [A] for Type I and Type IV pilus, respectively, facilitated de novo structural modelling for these filaments, revealing side chain structures in detail. Further, we resolved thousands of hydrated water molecules around and within the inner core of the filaments, functioning as a buffer to stabilise the otherwise metastable subunit assembly. The high-resolution structures offer novel insights into the subunit-subunit interactions, providing important clues to understand pilus assembly, stability, and flexibility.

microbiology↗

bioRxiv2023

A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries

Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a "host-guest" design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.

biochemistry↗

bioRxiv2021

Air plasma-activated medium exerts tumor-specific cytotoxicity via the oxidative stress-induced perinuclear mitochondrial clustering

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy eliminating cancers effectively and safely is the current clinical choice. Since aggressive tumors have inherent or acquired resistance to multidisciplinary therapies targeting apoptosis, tumor-specific induction of another cell death modality is a promising avenue to meet the goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can induce cell death in OS and OC via a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) because of the characteristic unipolar mitochondrial perinuclear aggregation. APAM had potent antitumor activity both in vitro and in vivo. APAM caused apoptosis, necrotic cell death, and autophagy. APAM contained hydrogen peroxide and increased mitochondrial ROS (mROS), while the antioxidant N-acetylcysteine (NAC) prevented cell death. MPMC occurred following mitochondrial fragmentation coinciding with nuclear damages. MPMC was accompanied by the tubulin network remodeling and mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC and the microtubule inhibitor, Nocodazole. Increased Cardiolipin (CL) oxidation was also seen, and NAC and the peroxy radical scavenger Ferrostatin-1 prevented it. In contrast, in fibroblasts, APAM induced minimal cell death, mROS generation, mLPO accumulation, CL oxidation, and MPMC. These results suggest that MPMC is a tumor-specific cause of cell death via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC might serve as a promising target for exerting tumor-specific cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/466636v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1118e2corg.highwire.dtl.DTLVardef@18cf99corg.highwire.dtl.DTLVardef@57a7edorg.highwire.dtl.DTLVardef@d1f447_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cancer biology↗

bioRxiv2020

Erythropoietin production?by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin {beta} pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

physiology↗