Biology subjects

Nishino, K.

Publications and source records attributed to Nishino, K..

14 recordsLinked to original sources

bioRxiv2026

Structural insights into fosfomycin efflux by a streptococcal ABC transporter

Gram-positive bacteria encode a broad array of ABC transporters that mediate substrate translocation across the cell membrane, with some contributing to their survival under environmental stresses such as antimicrobial exposure. While several of these transporters have been shown to exhibit multidrug efflux activity, the functional roles of many others remain unknown. Here, using an efflux pump screen in the opportunistic human pathogen Streptococcus pneumoniae, we identified a previously uncharacterized type IV ABC transporter (FoeAB) that confers resistance to the antibiotic fosfomycin. We show that purified FoeAB mediates fosfomycin transport in a liposome-reconstituted system and provide evidence that it functions as a multidrug efflux pump with substrate preferences distinct from those of known efflux pumps. Furthermore, we present cryogenic electron microscopy (cryo-EM) structures of FoeAB in inward- and outward-facing states, which reveal conformational changes associated with nucleotide binding and identify residues important for substrate transport. Collectively, these findings expand the known repertoire of antibiotic-exporting ABC transporters in Gram-positive bacteria and provide structural insight into its transport mechanism. SIGNIFICANCEEfflux pumps often play major roles in antibiotic resistance in clinically important pathogens, yet many of these transporters remain poorly defined. In this study, we identify and mechanistically characterize FoeAB, a previously unrecognized type IV ABC transporter in streptococcal species that mediates efflux of the antibiotic fosfomycin. By combining functional assays with structural studies of FoeAB in multiple conformational states, we propose a model for how this transporter recognizes its substrate and harnesses nucleotide-driven conformational changes to mediate substrate export. Our findings point to a previously underappreciated efflux capabilities in Gram-positive bacteria, highlighting the need for better characterization of membrane transporters to clarify their roles in bacterial physiology and drug resistance.

microbiology↗

bioRxiv2026

Fusion of a non-specific DNA-binding domain enhances Cas12a trans-cleavage for robust nucleic-acid diagnostics

CRISPR-Cas12a underlies powerful genome-editing and nucleic-acid detection technologies, yet its performance is limited by inefficient target engagement and low catalytic turnover, particularly at low target abundance and elevated temperatures. Here, we report a modular protein-engineering strategy to enhance Cas12a trans-activity by fusing the hyperthermophilic DNA-binding protein Sso7d to the N terminus of Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in kcat, and substantially accelerated trans-cleavage kinetics compared with wild-type Cas12a. These enhancements are observed across multiple guide RNAs, diverse DNA substrates, and a broad temperature range from 37 to 60 {degrees}C. The engineered Cas12a enables robust detection of the intrinsic blaOXA-51 and acquired blaOXA-24 antibiotic resistance genes from multiple Acinetobacter baumannii strains with independently validated resistance profiles, whereas wild-type Cas12a produces little or no detectable signal. Signal output is increased by up to [~]30-fold, expanding the effective detection window and shortening time-to-positive. Together, these results establish fusion of an accessory DNA-binding domain as a proof-of-concept for Sso7d fusion to LbCas12a and that generalization to other CRISPR effectors or non-specific DNA-binding domains represents an exciting avenue for future investigation.

biochemistry↗

bioRxiv2025

Linear ubiquitination triggers Amph-mediated T-tubule biogenesis

T-tubules are specialized invaginations of the plasma membrane essential for muscle contraction. While their physiological importance is well established, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis through proximity proteomics and RNAi screening in Drosophila. Loss of LUBEL leads to the formation of Amphiphysin (Amph)-positive membrane sheets instead of tubular networks in muscle cells. Mechanistically, the ubiquitin ligase activity of LUBEL, and direct interaction with Amph, a BAR domain protein involved in membrane tubule extension, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into condensates on membranes, facilitating Amph-mediated membrane tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across a broad range of species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover a previously unrecognized role for linear ubiquitination in membrane deformation driven by BAR domain proteins.

cell biology↗

bioRxiv2024

Functional dissection of metabolic trait-associated gene regulation in steady state and stimulated human skeletal muscle cells

Type 2 diabetes (T2D) is a common metabolic disorder characterized by dysregulation of glucose metabolism. Genome-wide association studies have defined hundreds of signals associated with T2D and related metabolic traits, predominantly in noncoding regions. While pancreatic islets have been a focal point given their central role in insulin production and glucose homeostasis, other metabolic tissues, including liver, adipose, and skeletal muscle, also contribute to T2D pathogenesis and risk. Here, we examined context-specific genetic regulation under basal and stimulated states. Using LHCN-M2 human skeletal muscle cells, we generated transcriptomic profiles and characterized regulatory activity of 327 metabolic trait-associated variants via a massively parallel reporter assay (MPRA). To identify condition-specific effects, we compared four different conditions: (1) undifferentiated, or (2) differentiated with basal media, (3) media supplemented with the AMP analog AICAR (to simulate exercise) or (4) media containing sodium palmitate (to induce insulin resistance). RNA-seq revealed these treatments extensively perturbed transcriptional regulation, with 498-3,686 genes showing significant differential expression between pairs of conditions. Among differentially expressed genes, we observed enrichment of relevant biological pathways including muscle differentiation (undifferentiated vs. differentiated), oxidoreductase activity (differentiated vs. AICAR), and glycogen binding (differentiated vs. palmitate). The results of our MPRA found broadly different levels of activity between all conditions. Our MPRA screen revealed a shared set of 7 variants with significant allelic activity across all conditions, along with a proportional number of variants showing condition-specific allelic bias and the total number of active oligos per condition. We found that a lead variant for serum triglyceride levels, rs490972, overlaps SP transcription factor motifs and has differential regulatory activity between conditions. Comparison of MPRA activity with paired gene expression data allowed us to predict that regulatory activity at this locus is mediated by SP1 transcription factor binding. While several of the MPRA variants have been previously characterized in other metabolic tissues, none have been studied in these stimulated states. Together, this work uncovers context-dependent transcriptomic and regulatory dynamics of T2D- and metabolic trait-associated variants in skeletal muscle cells, offering new insights into their functional roles in metabolic processes.

genomics↗

bioRxiv2024

Structural basis of nucleotide selectivity in pyruvate kinase

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

biochemistry↗

bioRxiv2024

The Rubicon-WIPI axis regulates exosome biogenesis during aging

Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.

cell biology↗

bioRxiv2024

Microbiome of psyllids of the family Aphalaridae, including Aphalara itadori, a potential biocontrol agent against Reynoutria spp.

Several European and North American countries have started releasing the Japanese knotweed psyllid Aphalara itadori (Hemiptera: Aphalaridae) to control the Japanese knotweed Reynoutria japonica (Caryophyllales: Polygonaceae) and its relatives, which are among the worst invasive exotic plants. However, establishing populations of the current Kyushu and Hokkaido strains in the field has not been successful, desiring new lineages. Moreover, little is known about the microbiome of the current strains, which potentially impacts properties as biocontrol agents. Hence, this study analyzed the microbiota of an A. itadori strain newly collected on Honshu Island, along with related species of the family Aphalaridae, using amplicon sequencing of 16S rRNA genes. The A. itadori symbionts were further located using fluorescence in situ hybridization. The results demonstrated that the analyzed A. itadori strain has a dual symbiotic system with "Candidatus Carsonella ruddii" (Gammaproteobacteria: Oceanospirillales) and Sodalis sp. (Gammaproteobacteria: Enterobacterales) harbored in the bacteriome, suggesting their evolutionarily stable mutualistic relationships with A. itadori. The central area of the bacteriome harboring Sodalis appeared to comprise uninucleate bacteriocytes with nuclei larger than those of bacteriocytes for Carsonella, rather than a syncytium with smaller nuclei as previously reported for various psyllid lineages. No known plant pathogens or manipulators of insect reproduction were identified in the analyzed strain, indicating its suitability as a biocontrol agent, posing a minimum risk to the ecosystem. Besides distinct Carsonella lineages, the analysis identified Sodalis independently acquired by Craspedolepta miyatakeai, and an ambiguous Enterobacterales symbiont in Epheloscyta kalopanacis. Only Carsonella was identified in Togepsylla matsumurana.

molecular biology↗

bioRxiv2023

Population-scale skeletal muscle single-nucleus multi-omic profiling reveals extensive context specific genetic regulation

Skeletal muscle, the largest human organ by weight, is relevant in several polygenic metabolic traits and diseases including type 2 diabetes (T2D). Identifying genetic mechanisms underlying these traits requires pinpointing cell types, regulatory elements, target genes, and causal variants. Here, we use genetic multiplexing to generate population-scale single nucleus (sn) chromatin accessibility (snATAC-seq) and transcriptome (snRNA-seq) maps across 287 frozen human skeletal muscle biopsies representing nearly half a million nuclei. We identify 13 cell types and integrate genetic variation to discover >7,000 expression quantitative trait loci (eQTL) and >100,000 chromatin accessibility QTLs (caQTL) across cell types. Learning patterns of e/caQTL sharing across cell types increased precision of effect estimates. We identify high-resolution cell-states and context-specific e/caQTL with significant genotype by context interaction. We identify nearly 2,000 eGenes colocalized with caQTL and construct causal directional maps for chromatin accessibility and gene expression. Almost 3,500 genome-wide association study (GWAS) signals across 38 relevant traits colocalize with sn-e/caQTL, most in a cell-specific manner. These signals typically colocalize with caQTL and not eQTL, highlighting the importance of population-scale chromatin profiling for GWAS functional studies. Finally, our GWAS-caQTL colocalization data reveal distinct cell-specific regulatory paradigms. Our results illuminate the genetic regulatory architecture of human skeletal muscle at high resolution epigenomic, transcriptomic, and cell-state scales and serve as a template for population-scale multi-omic mapping in complex tissues and traits.

genomics↗

bioRxiv2023

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Most genome-wide association signals for complex disease reside in the noncoding genome, where defining function is nontrivial. MPRAs (massively parallel reporter assays) offer a scalable means to identify functional regulatory elements, but are typically conducted without regard to cell type, pairing cloned fragments with a generic housekeeping promoter. To explore the context-sensitivity of MPRAs, we screened enhancer activity across a panel of nearly 12,000 198-bp fragments spanning over 300 type 2 diabetes- and metabolic trait-associated regions in the 832/13 rat insulinoma beta cell line, a relevant model of pancreatic beta cells. We explored these fragments context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin (INS) gene. We identified clear effects of MPRA construct design on enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. Promoter choice also influenced MPRA activity (n = 698/11,656), mostly biased towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory regions. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

genomics↗

bioRxiv2022

Role of the drug efflux pump in the intrinsic cefiderocol resistance of Pseudomonas aeruginosa

Cefiderocol is a novel siderophore cephalosporin antibiotic exhibiting activities against carbapenem-resistant Gram-negative bacteria including Pseudomonas aeruginosa and Enterobacteriaceae. Drug efflux pumps are reportedly involved in both intrinsic and acquired drug resistance, although their role in bacterial cefiderocol susceptibility remains poorly understood. In this study, we investigated how drug efflux pumps contribute to bacterial cefiderocol susceptibility using the efflux pump(s)-deficient and overexpressing strains of P. aeruginosa, Escherichia coli, and Salmonella enterica. We observed that the mexAB-oprM-deficient P. aeruginosa mutant displayed increased cefiderocol susceptibility compared to the wild-type strain. The overexpression of mexAB-oprM or mexXY-oprM in the mexAB-oprM-deficient mutant increased the MIC value of cefiderocol. Furthermore, the pump inhibitor phenylalanine-arginine {beta}-naphthylamide increased cefiderocol susceptibility in wild-type P. aeruginosa whereas it did not affect the susceptibility of the mexAB-oprM-deficient mutant. These data indicate that the MexAB-OprM drug efflux system contributes to the intrinsic cefiderocol resistance of P. aeruginosa. In addition, MexXY-OprM partially complemented the function of MexAB-OprM in the cefiderocol susceptibility, when expressed.

microbiology↗

bioRxiv2022

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the low abundance of protein biotinylation in cells required large amounts of cellular proteins to detect enough biotinylated peptides. Here we optimized the workflow for efficient enrichment and identification of biotinylated peptides. The most efficient recovery was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and the optimal temperature and salt concentration for elution. Using the optimized workflow, over 2-fold more biotinylated peptides were identified with higher purity from RAW264.7 macrophages expressing TurboID-fused STING. In addition, sequential digestion with Glu-C and trypsin led to the identification of biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon EGF stimulation. This workflow would be useful not only for BioID and cell surface proteomics but also for various other applications based on protein biotinylation.

biochemistry↗

bioRxiv2021

Proximal binding pocket Arg717 substitutions in Escherichia coli AcrB cause clinically relevant divergencies in resistance profiles

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 g/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and {beta}-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors. ImportanceThe over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as {beta}-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.

molecular biology↗

bioRxiv2021

Identification of bacterial drug-resistant cells by the convolutional neural network in transmission electron microscope images

The emergence of bacteria that are resistant to antibiotics is common in areas where antibiotics are used widely. The current standard procedure for detecting bacterial drug resistance is based on bacterial growth under antibiotic treatments. Here we describe the morphological changes in enoxacin-resistant Escherichia coli cells and the computational method used to identify these resistant cells in transmission electron microscopy (TEM) images without using antibiotics. Our approach was to create patches from TEM images of enoxacin-sensitive and enoxacin-resistant E. coli strains, use a convolutional neural network for patch classification, and identify the strains on the basis of the classification results. The proposed method was highly accurate in classifying cells, achieving an accuracy rate of 0.94. Using a gradient-weighted class activation mapping to visualize the region of interest, enoxacin-resistant and enoxacin-sensitive cells were characterized by comparing differences in the envelope. Moreover, Pearsons correlation coefficients suggested that four genes, including lpp, the gene encoding the major outer membrane lipoprotein, were strongly associated with the image features of enoxacin-resistant cells.

systems biology↗

bioRxiv2018

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.

cell biology↗