Shin-Ichiro Miyashita's research while affiliated with Boston Children's Hospital and other places

Publications (38)

Article
Full-text available
Botulinum neurotoxins (BoNTs) are the most potent toxins known and are also utilized to treat a wide range of disorders including muscle spasm, overactive bladder, and pain. BoNTs’ ability to target neurons determines their specificity, potency, and therapeutic efficacy. Homologous synaptic vesicle membrane proteins synaptotagmin-1 (Syt1) and synap...
Article
Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterror...
Article
Full-text available
Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A–G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-estab...
Article
Full-text available
Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here,...
Article
Full-text available
This data article provides atomic force microscopy (AFM) amplitude images of botulinum toxin complex (TC) molecules produced by Clostridium botulinum serotype D strain. C. botulinum produces different-sized TC molecules, such as a complex of botulinum neurotoxin and nontoxic nonhemagglutinin proteins (M-TC) and complex of M-TC and hemagglutinin sub...
Article
Full-text available
Potassium ion (K⁺) homeostasis and dynamics play critical roles in biological activities. Here we describe three genetically encoded K⁺ indicators. KIRIN1 (potassium (K) ion ratiometric indicator) and KIRIN1-GR are Förster resonance energy transfer (FRET)-based indicators with a bacterial K⁺ binding protein (Kbp) inserting between the fluorescent p...
Article
Botulinum neurotoxins (BoNTs) are the most potent toxin known to man and a significant threat as a weapon of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5–10% mortality rate. Existing treatment options cannot access or neutralize to...
Preprint
Full-text available
Potassium ion (K ⁺ ) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K ⁺ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K ⁺ in...
Article
Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocyto...
Article
Full-text available
De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation se...
Article
Full-text available
Botulinum neurotoxin (BoNT) associates with nontoxic proteins, either a nontoxic nonhemagglutinin (NTNHA) or the complex of NTNHA and hemagglutinin (HA), to form M- or L-toxin complexes (TCs). Single BoNT and NTNHA molecules are associated and form M-TC. A trimer of the 70-kDa HA protein (HA-70) attaches to the M-TC to form M-TC/HA-70. Further, 1-3...
Data
Peptide fragments of X-LC-HN under limited proteolysis analyzed by TMT labeling and quantitative mass spectrometry. His6-tagged recombinant X-LC-HN was labeled with the light TMT. Equal amounts of X-LC-HN samples were exposed to Lys-C and then labeled with the heavy TMT. Both samples were then digested with chymotrypsin, combined, and subjected to...
Article
Full-text available
Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a nove...
Article
Full-text available
The botulinum neurotoxin (BoNT) causes muscle paralysis and is the most potent toxin in nature. BoNT is associated with a complex of auxiliary “Non-Toxic” proteins, which constitute a large-sized toxin complex (L-TC). However, here we report that the “Non-Toxic” complex of serotype D botulinum L-TC, when administered to rats, exerts in-vivo toxicit...
Article
Full-text available
Clostridium botulinum strains produce a large-sized toxin complex (TC) that is composed of botulinum neurotoxin (BoNT), nontoxic non-hemagglutinin, and three different hemagglutinins (HA-70, HA-33, and HA-17). HA components enhance toxin delivery across the intestinal cell wall in a sugar chain-dependent manner. Here we characterized the sugar reco...
Article
Serotype D botulinum toxin (BoNT) complex (TC), a causative agent of foodborne botulism in animals, traverses the gastrointestinal tract and circulation, eventually becoming localized in neuromuscular junctions, where the serotype D BoNT cleaves SNARE substrate synaptobrevin II involved in neurotransmitter release. During this process, BoNT must pa...
Article
The large toxin complex (L-TC) produced by Clostridium botulinum is formed from the M-TC (BoNT/NTNHA complex) by conjugation of M-TC with HA-33/HA-17 trimer consists of two HA-33 proteins and a single HA-17 protein. This association is mediated by HA-70, which interacts with HA-17. The current study aims to identify the regions of the HA-70 molecul...
Article
Full-text available
Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monola...
Article
Botulinum neurotoxin (BoNT) associates with nontoxic nonhemagglutinin (NTNHA) yielding a complex in culture. BoNT and NTNHA have similar domain organizations, implying that they share common functions, although this remains unclear. Here we examined cell monolayer transport of serotype D NTNHA in the rat intestinal epithelial cell line IEC-6. NTNHA...
Article
Full-text available
Botulinum neurotoxin (BoNT) binds to nontoxic nonhemagglutinin (NTNHA) protein in a pH-dependent manner, and yields the protease-resistant BoNT/NTNHA complex. Here, we screened short peptides that bind to the serotype D NTNHA (NTNHA-D) using random phage display technique. NTNHA was fixed onto electrode of quartz crystal microbalance (QCM) apparatu...
Chapter
Full-text available
Botulinum neurotoxin (BoNT) produced by an anaerobic bacterium Clostridium botulinum causes highly fatal disease called botulism. BoNT is a zinc-dependent metalloprotease [1] with a molecular mass of 150 kDa, and classified into seven distinct serotypes A through G. Serotype A, B, E and F BoNTs dominantly cause human botulism, whereas serotype C an...
Article
Full-text available
The large-sized botulinum toxin complex (L-TC) is composed of botulinum neurotoxin (BoNT) and nontoxic proteins, e.g. nontoxic nonhemagglutinin (NTNHA) and three types of hemagglutinins (HAs; HA-33, HA-17 and HA-70). The nontoxic proteins play a critical role in L-TC oral toxicity by protecting the BoNT in the digestive tract, and facilitating abso...
Article
Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search...
Article
Full-text available
Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the pr...

Citations

... Thus, the flaccid paralysis of skeletal muscle appeared to be mainly dependent on the entry of BoNT/B into motor neurons by SytII whereas the entry of the toxin into parasympathetic neurons that innervate smooth muscle cells in the urinary bladder was predominantly mediated by SytI. If in humans a similar tissue specificity for BoNT/B uptake by SytI and SytII exists, the current assay might underestimate the potency for the action in skeletal muscle [28]. ...
... Moreover, the costs of the production and storage of large amounts of chimeric protein molecules are expected to be very high. However, the studies of McNutt et al. (2021) and Miyashita et al. (2021) provide a proof of principle of a novel therapeutic intervention of general value; one that can be extended to a variety of human diseases caused by pathogenic protein mutants present in the cytosol of neurons. ...
... This may partially explain why AnkG enrichment is selectively reduced without NF because of a mismatch in localization between AnkG and CK2-phosphorylated Na v . Future development of genetically encoded fluorescent Na + and K + indicators (Shen et al., 2018) to study channel function within the AIS as well as the continued development of super-resolution microscopy (Sigal et al., 2018) may help investigate this mechanism further. ...
... Interestingly, in E1 GBS, key interacting residues unique to BoNT/E have been identified, along with a significant rearrangement of loop 1228-1237, upon carbohydrate binding [42]. In BoNT/B, the LBL is located between the GBS and the receptor binding site, and is significantly exposed to the solvent; in particular the side-chains of W 1248 and W 1249 [43]. In the X-ray crystallographic structures of BoNTs, the regions LBL and GBS are close in 3D space, and correspond to well-defined binding regions (see, e.g., Figure S2 for A1 in the open state; PDB entry: 3BTA). ...
... Surface sGAG is essential for LDLR/LRP1/Megalin-mediated uptake of Tcnα. Cell-surface sGAG can mediate the attachment of Tcnα and Clostridioides difficile toxin A (TcdA) and allow them to be enriched on the cell surface 13,27 . To demonstrate the sGAG-binding potentials of other major LCTs, we performed the heparin-beads pulldown experiment with the purified LCT proteins. ...
... The affinity (K d ) of purified GINKO2 for K + is 15.3 mM. While GINKO1 shows substantial sodium (Na + )-dependent fluorescence response at concentrations below 150 mM, complicating applications where Na + is abundant [13], GINKO2 is not responsive to Na + at concentrations up to 150 mM, thus showing an improved specificity (Fig 3D). As the affinity for K + of GINKO2 (15.3 mM) is substantially lower than that of GINKO1 (0.42 mM) (S3 Table), the affinity for Na + may have also decreased Green-colored residues are on GFP, orange-colored residues are on Kbp, gray colored residues are on linkers, and blue-colored L and magenta-colored E (N in GINKO1.2) are the positions of "gatepost" residues that define the optimal insertion points in EGFP [22]. ...
... In general, up to the present time, inhibitors of the metalloprotease activity of BoNTs were found to be effective only at levels (A) and/or (B) with K i or IC 50 values in the 10 -6 -10 −8 Molar range. (Caglič et al. 2014;Kumar et al. 2016;Bremer et al. 2017;Vieni et al. 2018;Patel et al. 2018;Garland et al. 2019;Amezcua et al. 2021;Lin et al. 2021;Turner et al. 2021a, b). None the less, these studies are very important because they provide the molecular basis to design novel and specific inhibitors of the BoNT active site acting in cultured neurons in the nanomolar range, a value which is a prerequisite figure to proceed to animal studies (level C). ...
... BoNT/X was demonstrated to cleave multiple VAMPs including several non-canonical substrates that cannot be cleaved by any other BoNTs. A year later, another BoNT-like toxin was discovered within a strain of commensal organism, Enterococcus faecium [15,16]. This BoNT, labeled BoNT/En, was shown to cleave both SNAP25 and VAMP2 at unique sites, and reside on a common conjugative plasmid, suggesting a mode of circulation within Enterococcus strains. ...
... Indeed, we can easily find multiple promising nanobodies for endogenous proteins in an open website database 55 . In addition, rapid approaches to generate large repertoires of recombinant nanobodies and de novo proteins designed for creating customized small binding proteins have been reported 31,56 . A recent paper also showed that machine learning using only structural information of a target protein can produce specific small-sized specific binding proteins 57 . ...
... Genome analysis showed the presence of a novel toxinotype called BoNT/X in a C. botulinum type B, which also produces BoNT/B2. BoNT/X retains a low sequence identity with other types and is not recognized by antibodies against these (Zhang et al. 2017). bont related sequences identified in various non-clostridial species have not been involved in clinical botulism such as Weisenella oryzae (BoNT/Wo or BoNT/I) from fermented rice, Chryseobacterium piperi (Cp1) from sediment, an Enterococcus faecalis strain (BoNT/J, or BoNT/En, or eBoNT/J) isolated from a cow without clinical symptom of botulism, and Paraclostridium bifermentans (paraclostridial mosquitocidal protein 1, PMP1; Rasetti-Escargueil and Popoff 2020; Table 30.1), ...