Biology subjects

Kumeta, H.

Publications and source records attributed to Kumeta, H..

10 recordsLinked to original sources

bioRxiv2026

Calibrated ADAMTS13 inhibition prevents cardiovascular shear coagulopathy

Mechanical circulatory support essential for managing severe heart failure frequently triggers bleeding complications, driven by shear stress-induced over-proteolysis of von Willebrand factor (VWF) by ADAMTS13. Inhibiting ADAMTS13 presents a rationale to treat this condition, known as acquired von Willebrand syndrome (AVWS). However, conventional therapeutic strategies remain limited due to the risk of triggering thrombotic thrombocytopenic purpura. Here we show that HA10, a humanized anti-ADAMTS13 antibody, preserves a residual level of ADAMTS13 activity above the thrombosis-associated threshold. Multimodal structural and biophysical analyses--including NMR, SAXS, and SANS--revealed that HA10 bound to the disintegrin-like domain of ADAMTS13, dynamically competing with VWF while leaving 10- 20% residual enzymatic activity. The therapeutic efficacy and safety of HA10 were verified in non-human primate models of AVWS. Our findings establish a novel paradigm of enzymatic calibration rather than complete blockade, offering a mechanistically targeted and safe therapeutic approach for cardiovascular bleeding.

biochemistry↗

bioRxiv2025

OptoChaperone - A biohybrid tool for regulating protein condensates in cells and in vitro

Protein condensates formed via liquid-liquid phase separation (LLPS) are increasingly recognized as key players in diverse cellular processes, including those associated with disease. Despite extensive efforts to characterize their formation and function, tools that enable precise, reversible, and spatiotemporal control of LLPS remain limited. Here, we report OptoChaperone, a light-activatable molecular system designed to manipulate protein condensates both in vitro and in living cells. This biohybrid system leverages photoresponsive switching to control chaperone activity: blue light triggers the suppressive function, leading to the dissolution of protein condensates, whereas UV light deactivates the system, allowing condensate formation. We demonstrate the efficacy of OptoChaperone in regulating several disease-related protein condensates, such as fused in sarcoma, TAR DNA-binding protein 43, and heat shock factor 1. Importantly, the system exhibits reversible and robust control over droplet dynamics without requiring chemical additives or genetic modifications of the client proteins. Given the reversibility and efficiency of OptoChaperone in the manipulation of protein condensates, this tool offers a powerful platform for dissecting the roles of protein condensation in cellular physiology and pathology. This strategy also holds potential for broader applications in synthetic biology, biomolecular engineering, and therapeutic modulation of aberrant phase separation.

biochemistry↗

bioRxiv2025

A Disease-Associated Mutation Impedes PPIA Through Allosteric Dynamics Modulation

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron degeneration. Peptidylprolyl cis-trans isomerase A (PPIA) is a molecular chaperone involved in protein folding, and its dysfunction has been linked to ALS pathogenesis as proline is recognized as a key residue for maintaining proper folding of ALS-related proteins. A recent study identified a K76E mutation in PPIA in sporadic ALS patients, but its effects on protein function and structure remain unclear. In this study, we used biochemical and biophysical techniques to investigate the structural and functional consequences of the K76E mutation. Our results show that K76E significantly reduces enzyme activity without affecting structure, monodispersity, or substrate recognition. Significant effects of K76E mutation were identified by relaxation dispersion NMR experiments, showing that K76E disrupts key protein dynamics and alters an allosteric network essential for isomerase activity. Corroborated by theoretical kinetic analysis, this dynamics data, revealing the exchange process for K76E approximately one-tenth that of the wild type, explain the reduced cis-trans isomerase activity of the K76E mutant. These findings suggest that the pathogenic effect of K76E arises primarily from impaired protein dynamics rather than direct structural disruption. Our study provides new insights into the molecular mechanisms underlying ALS-associated mutations and their impact on protein function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/653011v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@82f81corg.highwire.dtl.DTLVardef@6f0a04org.highwire.dtl.DTLVardef@1fabe12org.highwire.dtl.DTLVardef@d49d11_HPS_FORMAT_FIGEXP M_FIG For Table of Contents C_FIG

biochemistry↗

bioRxiv2024

Structural basis for phosphatidylcholine synthesis by bacterial phospholipid N-methyltransferases

In phosphatidylcholine (PC)-containing bacteria, PC is synthesized by phospholipid N-methyltransferases (Pmts) and plays an important role in the interactions between symbiotic and pathogenic bacteria and their eukaryotic host cells. Pmts catalyze the SAM dependent three methylation reactions of the head group of phosphatidylethanolamine (PE) to form PC through monomethyl PE and dimethyl PE. However, the precise molecular mechanisms underlying PC biosynthesis by PmtA remain largely unclear, owing to the lack of structural information. Here, we determined the crystal structures of Agrobacterium tumefaciens Pmt (AtPmtA) in complex with SAH or 5'-methylthioadenosine. Crystal structures and NMR analysis revealed the binding mode of AtPmtA to SAH in solution. Structure-based mutational analyses showed that a conserved tyrosine residue in the substrate-binding groove is involved in methylation. Furthermore, we showed that differences in substrate specificity among Pmt homologs were determined by whether the amino acid residues comprising the substrate-binding groove were isoleucine or phenylalanine. These findings provide a structural basis for understanding the mechanisms underlying Pmts-mediated PC biosynthesis.

biochemistry↗

bioRxiv2024

Phase separation promotes Atg8 lipidation for autophagy progression

Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the pre-autophagosomal structure (PAS) in yeast, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS as a liquid droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets via specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution experiments reveal that E3-enriched early PAS droplets promote Atg8 lipidation and incorporate Atg8-coated vesicles to the interior, thereby protecting them from Atg4-mediated delipidation. These data suggest that the PAS utilizes its liquid-like property to function as an efficient production site for lipidated Atg8 and pool membrane seeds to drive autophagosome formation.

cell biology↗

bioRxiv2024

Ca2+-driven PDIA6 phase separation to ensure proinsulin quality control

The endoplasmic reticulum (ER) plays key roles in protein quality control1,2 and dynamic Ca2+ storage3,4 in eukaryotic cells. However, the protein homeostasis (proteostasis) system that regulates these ER functions is still incompletely characterised. Previous study revealed the importance of oligomerization in the function PDIA1, an ER-resident disulfide isomerase and molecular chaperone, regulates oligomeric states in accordance with client folding5. This result suggests that at least some of the 20 members of other PDI family may undergo regulated self-assembly in order to optimally function. Here, we show that Ca2+ triggers the phase separation of PDIA6 into liquid-like condensates. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the Ca2+-driven condensation of PDIA6 recruits PDIA3 and proinsulin, thus increasing their local concentrations. This process results in the 30-fold enhancement of proinsulin folding and in the inhibition of proinsulin aggregation. Our findings shed light on a condensation-driven Ca2+-mediated proteostasis cascade in the ER by revealing how the efficiency of the protein folding process can be enhanced within quality control granules.

biochemistry↗

bioRxiv2024

Conserved loop of a phase modifier endows protein condensates with fluidity

Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion in C9orf72 trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherin{beta}2 (Kap{beta}2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kap{beta}2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kap{beta}2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kap{beta}2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight into C9orf72-ALS related to age and repeat expansion.

biochemistry↗

bioRxiv2024

Client recognition differences between PDI and ERp46 to guide oxidative folding

Endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) family members function not only as disulfide bond-catalysts but also as chaperones for the oxidative folding of client proteins. However, due to the scarcity of structural data, the client recognition mechanism remains poorly understood. We report the distinct recognition mechanisms for PDI/ERp46 proteins to recruit a reduced and denatured bovine pancreatic trypsin inhibitor (BPTI). NMR data demonstrated that PDI recognizes a broad region consisting of [~]30 amino acid residues in unfolded BPTI in order to function as a chaperone, while ERp46 interacts nonspecifically and weakly with unfolded BPTI to promiscuously and rapidly introduce disulfide bonds. These two distinct client recognition modes are likely important for the functional modulation of the disulfide-catalyst and chaperone against client proteins. Client recognition differences between PDI family proteins likely contribute to determining whether disulfide bonds are introduced into the client protein or aggregates are reduced to an unfolded state during the early folding step. Thus, the mammalian ER ensures concerted protein quality control through the differences in the client recognition modes of PDI family proteins, resulting in the production of large quantities of multiple-disulfide bonded proteins.

biochemistry↗

bioRxiv2023

Zinc finger domains bind low-complexity domain polymers

Self-association of low-complexity protein sequences (LC domains) is important for polymer formation. Several molecular chaperones are involved in the regulation of LC domain polymer formation. However, the mechanisms underlying cell recognition of LC domain polymers remain unclear. Here we show that zinc finger domains (ZnFs) bind LC domains of RNA-binding proteins in a cross-{beta} polymer-dependent manner. ZnFs bound to LC domain hydrogels and suppressed LC domain polymer formation. Moreover, ZnFs preferentially recognize LC domains in the polymeric state. These findings suggest that ZnFs act as physiological regulators of LC domain polymer formation.

biochemistry↗

bioRxiv2020

A unique adhesive motif of protein disulfide isomerase P5 supports its function via dimerization

P5, also known as PDIA6, is a PDI-family member that plays an important role in the ER quality control. Herein, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. This motif is apparently similar to, but radically different from conventional leucine-zipper motifs, in that the former includes a periodic repeat of leucine or valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel -helices, unlike the latter of which the leucine residues appear every two helical turns on [~]30-residue parallel -helices at dimer interfaces. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as an aberrant protein that induces the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1 via reduction of intermolecular disulfide bonds and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and physiological function of P5.

biochemistry↗