Biology subjects

Fujita, J.

Publications and source records attributed to Fujita, J..

10 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↗

bioRxiv2025

Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region

Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.

biochemistry↗

bioRxiv2024

Structural basis for the interaction between the bacterial cell division proteins FtsZ and ZapA

Cell division in most bacteria is regulated by the tubulin homolog FtsZ as well as ZapA, a FtsZ-associated protein. However, how FtsZ and ZapA function coordinately has remained elusive. Here we report the cryo-electron microscopy structure of the ZapA-FtsZ complex. The complex forms an asymmetric ladder-like structure, in which the double antiparallel FtsZ protofilament on one side and a single protofilament on the other side are tethered by ZapA tetramers. In the complex, the extensive interactions of FtsZ with ZapA cause a structural change of the FtsZ protofilament, and the formation of the double FtsZ protofilament increases electrostatic repulsion. High-speed atomic force microscopy analysis revealed cooperative interactions of ZapA with FtsZ at a molecular level. Our findings not only provide a structural basis for the interaction between FtsZ and ZapA but also shed light on how ZapA binds to FtsZ protofilaments without disturbing FtsZ dynamics to promote cell division.

molecular biology↗

bioRxiv2024

Dimeric assembly of F1-like ATPase for Mycoplasma gliding motility

Rotary ATPases, including F1Fo- and V/A-ATPases, are molecular motors that exhibit rotational movements for energy conversion1. In the gliding bacterium, Mycoplasma mobile, a dimeric F1-like ATPase forms a chain structure with the glycolytic enzyme, phosphoglycerate kinase (PGK), within the cell2, which is proposed to drive the bacterial gliding motility2-4. However, the mechanisms of force generation and transmission remain unclear. Here, we present a 3.2 [A] resolution structure of the dimeric ATPase complex obtained using electron cryomicroscopy (cryo-EM). Notably, the structure revealed an assembly distinct from that of known dimeric forms of F1Fo-ATPase5, despite containing conserved F1-ATPase structures. The two ATPase units were interconnected by GliD dimers, previously identified as MMOB16202,6. Gli{beta}, a homologue of the F1-ATPase catalytic subunit6, exhibited a specific N-terminal region that incorporates PGK into the complex. Structural conformations of the catalytic subunits, catalytically important residues, and nucleotide-binding pattern of the catalytic sites of the ATPase displayed strong similarities to F1-ATPase, suggesting a rotation based on the rotary catalytic mechanism conserved in rotary ATPases1,7-10. Overall, the cryo-EM structure underscores an evolutionary connection in the rotary ATPases and provides insights into the mechanism through which F1-like ATPase drives bacterial gliding motility.

microbiology↗

bioRxiv2023

Internal Translation of p53 Oncoproteins During Integrated Stress Response Confers Survival Advantage on Cancer Cells

p53 is the most known and studied tumour suppressor gene. Yet we have recently shown that p53 is also a proto-oncogene, as it encodes the {Delta}160p53 oncoprotein. Integrated stress response (ISR) is a survival pathway frequently activated in cancers, marked by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2) and a defined reprogramming in mRNA translation. Here we identified ISR as a powerful trigger of p53 oncogene, leading to the induction of not only {Delta}160p53 but also {Delta}133p53, another protein variant of the p53 gene. Upon ISR the two isoforms were translated internally from p53 full-length (FL) transcript through an internal regulator of expression site (IRES) located in the vicinity of codon 160. Frameshift mutations upstream of codons 133 and 160 demonstrated that FLp53 protein synthesis is not required for making {Delta}133p53 and {Delta}160p53. Instead, targeting IRES(160) with an antisense oligo was sufficient to efficiently and specifically impair the expression of these isoforms without affecting FLp53 levels. This in turn averted ISRs protective program culminating in cancer cell cycle arrest and death. Mechanistically, FLp53 showed 3 times more affinity to {Delta}160p53 than to other isoforms, {Delta}40p53 or {Delta}133p53. During ISR {Delta}160p53 localized to the nucleus and strongly inhibited FLp53-mediated activation of pro-apoptotic gene p53 upregulated modulator of apoptosis (PUMA). Our results uncover a new branch of the ISR network essential for cancer cell survival and growth and establish the proof of concept for a new strategy to target cancer.

cancer biology↗

bioRxiv2022

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by epitope selection

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

biochemistry↗

bioRxiv2022

High-resolution structure of a microtubule-like tube composed of FtsZ-monobody complexes

FtsZ, a bacterial tubulin homologue, forms protofilaments and the Z-ring, which acts as a scaffold for accessory proteins during cell division. Although various studies have revealed its molecular mechanisms, the lack of high-resolution solution structures has hindered the understanding of the detailed mechanisms. Here, we developed a monobody (Mb) that binds FtsZs from Escherichia coli and Klebsiella pneumoniae (KpFtsZ) without affecting their GTPase activities. When expressed in E. coli cells, the Mb did not inhibit Z-ring formation but did inhibit cell division. The crystal structures of the KpFtsZ-Mb complexes revealed the epitope, and the cryoEM structure at 2.67 [A] resolution showed a double helical tube consisting of two KpFtsZ protofilaments stabilized by the Mb filling interfilament gaps. Our structural analyses highlight the similarity between the microtubule and the FtsZ tube and the importance of the plasticity of FtsZ protofilaments.

molecular biology↗

bioRxiv2022

Onecut1 partially contributes to liver progenitor cell transition and acquisition of metastatic potential in hepatocellular carcinoma.

Metastasis-initiating cells are considered to originate from stem cell-like cancer cells. In hepatocellular carcinoma, liver progenitor-like cells are reported to be derived from hepatocytes, indicating the possible acquisition of metastatic potential during hepatocyte-to-cholangiocyte transdifferentiation. Consistent with the expression pattern observed during ductal plate formation, we revealed an LPC transition with Onecut1 accumulation both during hepatocyte-to-cholangiocyte transdifferentiation and in a cell model. This event may be associated with transient acquisition of metastatic potential.

cancer biology↗

bioRxiv2021

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2* and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 [A] resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

molecular biology↗

bioRxiv2021

Nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants

We are in the midst of the historic coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Although countless efforts to control the pandemic have been attempted--most successfully, vaccination1-3--imbalances in accessibility to vaccines, medicines, and diagnostics among countries, regions, and populations have been problematic. Camelid variable regions of heavy chain-only antibodies (VHHs or nanobodies)4 have unique modalities: they are smaller, more stable, easier to customize, and, importantly, less expensive to produce than conventional antibodies5, 6. We present the sequences of nine alpaca nanobodies that detect the spike proteins of four SARS-CoV-2 variants of concern (VOCs)--namely, the alpha, beta, gamma, and delta variants. We show that they can quantify or detect spike variants via ELISA and lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays7. The panel of nanobodies broadly neutralized viral infection by pseudotyped SARS-CoV-2 VOCs. Structural analyses showed that a P86 clone targeted epitopes that were conserved yet unclassified on the receptor-binding domain (RBD) and located inside the N-terminal domain (NTD). Human antibodies have hardly accessed both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins undetected by conventional antibodies and maintains activity against spike proteins carrying escape mutations.

molecular biology↗