Biology subjects

Kimoto, M.

Publications and source records attributed to Kimoto, M..

6 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into multiple BAM-bound conformations of BepA enabling substrate triage at the outer membrane

The outer membrane (OM) of Gram-negative bacteria acts as a selective permeability barrier against toxic compounds, and for this function, requires proper assembly of outer membrane proteins (OMPs) by the {beta}-barrel assembly machinery (BAM) complex. A periplasmic metalloprotease BepA promotes maturation of LptD, an essential OMP, while degrades LptD intermediates aberrantly stalled at BAM. However, how BepA switches between these functions remains unclear. Here, we report cryo-EM structures of BAM- BepA complexes that capture multiple BepA conformations. In vivo crosslinking and cysteine-accessibility analyses show that possible regulatory elements, 6- and 9-loops that cover the proteolytic active site, can assume open conformations in living cells. Functional analyses reveal that 6-loop opening supports substrate interaction, degradation, and membrane association, whereas 9-loop opening is required for proteolytic activation and stabilizes BAM association. These findings provide insights into how distinct loop rearrangements regulate BepA-mediated substrate triage at BAM.

molecular biology↗

bioRxiv2025

Structural basis of lipopolysaccharide translocon assembly mediated by the small lipoprotein LptM

Gram-negative bacteria possess an outer membrane (OM) that acts as a barrier against toxic compounds. Lipopolysaccharide (LPS) in the outer leaflet of the OM is crucial for barrier function. After its synthesis in the cytoplasm and translocation to the periplasm, LPS is transported to the OM by the LPS transport system. The LPS translocon, composed of an OM protein LptD and a lipoprotein LptE, mediates LPS assembly into the OM. Recently, the small lipoprotein LptM (YifL) was identified as a novel LptD/E-interactor that facilitates LptD maturation. However, its mechanism remains unclear. Here, we investigated the detailed interaction between LptM and LptD. We found that LptM interacts with the folded LptD intermediate at the late stage of its maturation. Mutational analyses demonstrated that the N-terminal conserved region (C20GLKGPLYF28) of LptM is essential for its function. Cryo-EM structural analysis of the E. coli LptD/E/M complex, combined with biochemical analyses, revealed the molecular basis of the LptM-LptD interaction and its functional importance. Thus, we propose that LptM functions as a "barrel-rivet," stabilizing LptD for its proper assembly.

molecular biology↗

bioRxiv2024

Direct high-throughput deconvolution of unnatural bases via nanopore sequencing and bootstrapped learning

The discovery of non-canonical bases (NCBs) in viruses and the development of synthetic xeno-nucleic acids (XNAs) to expand the genetic alphabet has spawned interest in many applications, from viral genomics, to synthetic biology and DNA storage. However, the inability to read non-canonical bases in a direct, high-throughput manner has been a significant limitation to its study and applicability. Here we demonstrate that XNA templates containing non-canonical bases can be directly and robustly sequenced (>2.3 million reads/flowcell, similar to DNA controls) on a MinION sequencer from Oxford Nanopore Technologies to obtain signal data that is significantly distinct from DNA controls (median fold-change >6x). To enable training of machine learning models that deconvolve these signals and basecall non-canonical and canonical bases, we developed a framework to synthesize a complex pool of 1,024 NCB-containing oligonucleotides with diverse 6-mer sequence contexts and high purity (>90% NCB-insertion on average). Bootstrapped models to assist in data preparation, and data augmentation with spliced reads to provide high context diversity, enabled learning of a generalizable model to call canonical as well as non-canonical bases with high accuracy (>80%) and specificity (99%). These results highlight the versatility of nanopore sequencing as a platform for interrogating nucleic acids for viral genomic and xenobiology applications, and the potential to transform the study of genetic material beyond those that use canonical bases.

synthetic biology↗

bioRxiv2022

Success probability of high-affinity DNA aptamer generation by genetic alphabet expansion

SummaryNucleic acid aptamers as antibody alternatives bind specifically to target molecules. These aptamers are generated by isolating candidates from libraries with random sequence fragments, through an evolutionary engineering system. We recently reported a high-affinity DNA aptamer generation method that introduces unnatural bases (UBs) as a fifth letter into the library, by genetic alphabet expansion. By incorporating hydrophobic UBs, the affinities of DNA aptamers to target proteins are increased over 100-fold, as compared to those of conventional aptamers with only the natural four letters. However, there is still plenty of room for improvement of the methods for routinely generating high-affinity UB-containing DNA (UB-DNA) aptamers. The success probabilities of the high-affinity aptamer generation depend on the existence of the aptamer candidate sequences in the initial library. We estimated the success probabilities by analysing several UB-DNA aptamers that we generated, as examples. In addition, we investigated the possible improvement of conventional aptamer affinities by introducing one UB at specific positions. Our data revealed that UB-DNA aptamers adopt specific tertiary structures, in which many bases including UBs interact with target proteins for high affinity, suggesting the importance of the UB-DNA library design.

synthetic biology↗

bioRxiv2022

Cardiac hemodynamics and ventricular stiffness of sea-run cherry salmon (Oncorhynchus masou masou) differ critically from those of landlocked masu salmon

Ventricular diastolic mechanical properties are important determinants of cardiac function and are optimized by changes in cardiac structure and physical properties. Oncorhynchus masou masou is an anadromous migratory fish of the Salmonidae family, and several ecological studies on it have been conducted; however, the cardiac functions of the fish are not well known. Therefore, we investigated ventricular diastolic function in landlocked (masu salmon) and sea-run (cherry salmon) types at 29-30 months post fertilization. Pulsed-wave Doppler echocardiography showed that the atrioventricular inflow waveforms of cherry salmon were biphasic with early diastolic filling and atrial contraction, whereas those of masu salmon were monophasic with atrial contraction. In addition, end-diastolic pressure-volume relationship analysis revealed that the dilatability per unit myocardial mass of the ventricle in cherry salmon was significantly suppressed compared to that in masu salmon, suggesting that the ventricle of the cherry salmon was relatively stiffer (relative ventricular stiffness index; p = 0.0263). Contrastingly, the extensibility of cardiomyocytes, characterized by the expression pattern of Connectin isoforms in their ventricles, was similar in both types. Histological analysis showed that the percentage of the collagen accumulation area in the compact layer of cherry salmon increased compared with that of the masu salmon, which may contribute to ventricle stiffness. Although the heart mass of cherry salmon was about 11-fold greater than that of masu salmon, there was no difference in the morphology of the isolated cardiomyocytes, suggesting that the heart of the cherry salmon grows by cell division of cardiomyocytes, but not cell hypertrophy. The cardiac physiological function of the fish varies with differences in their developmental processes and life history. Our multidimensional analysis of the O. maosu heart may provide a clue to the process by which the heart acquires a biphasic blood-filling pattern, i.e., a ventricular diastolic suction.

physiology↗

bioRxiv2021

Transient induction of cell cycle promoter Fam64a improves cardiac function through regulating Klf15-dependent cardiomyocyte differentiation in mice

The introduction of fetal or neonatal signatures such as cell cycle promoting genes into damaged adult hearts has been vigorously pursued as a promising strategy for stimulating proliferation and regeneration of adult cardiomyocytes, which normally cannot divide. However, cell division of cardiomyocytes requires preceding dedifferentiation with sarcomere disassembly and calcium dysregulation, which, in principle, compromises contractile function. To overcome this intrinsic dilemma, we explored the feasibility of optimizing the induction protocol of the cell cycle promoter in mice. As a model of this approach, we used Fam64a, a fetal-specific cardiomyocyte cell cycle promoter that we have recently identified. We first analyzed transgenic mice maintaining long-term cardiomyocyte-specific expression of Fam64a after birth, when endogenous expression was abolished. Despite having an enhanced proliferation of postnatal cardiomyocytes, these mice showed age-related cardiac dysfunction characterized by sustained cardiomyocyte dedifferentiation, which was reminiscent of the dilemma. Mechanistically, Fam64a inhibited glucocorticoid receptor-mediated transcriptional activation of Klf15, a key regulator that drives cardiomyocyte differentiation, thereby directing cardiomyocytes toward immature undifferentiated states. In contrast, transient induction of Fam64a in cryoinjured wildtype adult mice hearts improved functional recovery with augmented cell cycle activation of cardiomyocytes. These data indicate that optimizing the intensity and duration of the stimulant to avoid excessive cardiomyocyte dedifferentiation could pave the way toward developing efficient strategy for successful heart regeneration.

physiology↗