Biology subjects

Konuma, T.

Publications and source records attributed to Konuma, T..

5 recordsLinked to original sources

bioRxiv2026

Fine-Tuning α-Synuclein Phase Separation through Sequence-Optimized Peptide Modulators

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins underlies the formation of biomolecular condensates that regulate diverse cellular processes, while its dysregulation contributes to protein aggregation and disease. Despite its importance, molecularly defined and target-specific strategies to control LLPS remain limited. Here, we present a systematic framework for designing de novo peptides that induce and modulate LLPS of -synuclein. By integrating deep mutational scanning with peptide screening, we identified sequence features that govern condensate formation and enabled the creation of optimized peptides with high efficiency and specificity. Biophysical analyses revealed that LLPS efficiency is dictated by the interplay of solubility, multivalency, and cooperative interactions, resulting in a distinctive bell-shaped phase diagram. Thermodynamic measurements and imaging-based analyses further demonstrated that condensate stability and material properties can be rationally tuned through peptide optimization. Together, these findings establish generalizable design principles for engineering LLPS modulators in biologically and pathologically relevant protein systems.

biophysics↗

bioRxiv2024

Analysis of the high-order conformational changes in glyceraldehyde-3-phosphate dehydrogenase induced by nicotinamide adenine dinucleotide, adenosine triphosphate, and oxidants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis. As a moonlighting protein, GAPDH interacts with other molecules, seemingly unrelated to glycolysis, to exert additional functions, such as inducing apoptosis. However, specific mechanisms underlying these interactions remain unclear. Therefore, in this study, we aimed to elucidate these mechanisms by analyzing human and porcine GAPDHs based on their three-dimensional (3D) structures using various biophysical techniques, including nuclear magnetic resonance spectroscopy, gel filtration chromatography, and thermal shift assay. Although GAPDH became unstable when nicotinamide adenine dinucleotide (NAD+) was removed (apo state), the 3D structure of the tetramer was maintained regardless of the temperature. However, in the presence of adenosine triphosphate (ATP), GAPDH split into dimers at low temperatures, exposing the interface between the dimers and increasing the flexibility of the side chains at the site. Moreover, subunit splitting also occurred upon exposure to oxidizing agents, such as hydrogen peroxide. These findings suggest that GAPDH maintains a stable tetramer in the presence of NAD+ but becomes unstable and easily oxidized upon NAD+ depletion. When multiple residues, including those other than the cysteine residue at the active site, are oxidized by reactive oxygen species or nitric oxide, or when it interacts with ATP, GAPDH splits into dimers. This subunit splitting may trigger interactions with other molecules.

biochemistry↗

bioRxiv2024

Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3

DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique -helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

biochemistry↗

bioRxiv2023

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn2+-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

biochemistry↗

bioRxiv2022

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced - helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

biochemistry↗