| 1. |
- Gregg, Brieana M., et al.
(författare)
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Botulinum neurotoxin X lacks potency in mice and in human neurons
- 2024
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Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 15:3
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Tidskriftsartikel (refereegranskat)abstract
- Botulinum neurotoxins (BoNTs) are a class of toxins produced by Clostridium botulinum (C. botulinum) and other species of Clostridia. BoNT/X is a putative novel botulinum neurotoxin identified through genome sequencing and capable of SNARE cleavage, but its neurotoxic potential in humans and vertebrates remained unclear. The C. botulinum strain producing BoNT/X, Strain 111, encodes both a plasmid-borne bont/b2 as well as the chromosomal putative bont/x. This study utilized C. botulinum Strain 111 from Japan as well as recombinantly produced full-length BoNT/X to more fully analyze this putative pathogenic toxin. We confirmed production of full-length, catalytically active native BoNT/X by C. botulinum Strain 111, produced as a disulfide-bonded dichain polypeptide similar to other BoNTs. Both the purified native and the recombinant BoNT/X had high enzymatic activity in vitro but displayed very low potency in human-induced pluripotent stem cell-derived neuronal cells and in mice. Intraperitoneal injection of up to 50 µg of native BoNT/X in mice did not result in botulism; however, mild local paralysis was observed after injection of 2 μg into the gastrocnemius muscle. We further demonstrate that the lack of toxicity by BoNT/X is due to inefficient neuronal cell association and entry, which can be rescued by replacing the receptor binding domain of BoNT/X with that of BoNT/A. These data demonstrate that BoNT/X is not a potent vertebrate neurotoxin like the classical seven serotypes of BoNTs.
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| 2. |
- Kinsolving, Julia, et al.
(författare)
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Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7
- 2024
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Ingår i: Cell Reports. - 2211-1247. ; 43:2
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Tidskriftsartikel (refereegranskat)abstract
- The G protein -coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live -cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA -approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore -forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
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| 3. |
- Košenina, Sara, 1993-, et al.
(författare)
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The cryo-EM structure of the BoNT/Wo-NTNH complex reveals two immunoglobulin-like domains
- 2024
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Ingår i: The FEBS Journal. - 1742-464X .- 1742-4658. ; 291:4, s. 676-689
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Tidskriftsartikel (refereegranskat)abstract
- The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like (Big) domains on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT–NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.
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| 4. |
- Real, Karine Queiroz Zetune Villa, et al.
(författare)
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A Versatile Synaptotagmin-1 Nanobody Provides Perturbation-Free Live Synaptic Imaging And Low Linkage-Error in Super-Resolution Microscopy
- 2023
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Ingår i: Small Methods. - 2366-9608. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy alters the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here a nanobody is presented that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions as an intrabody (iNbSyt1) in living neurons and is minimally invasive, leaving synaptic transmission almost unaffected, as suggested by the crystal structure of the NbSyt1 bound to Synaptotagmin-1 and by the physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as showcased here by measuring spatially localized presynaptic Ca2+ with a NbSyt1- jGCaMP8 chimera. Moreover, the small size of NbSyt1 makes it ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience with unprecedented precision across multiple spatiotemporal scales.
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| 5. |
- Stenmark, Pål, 1976-
(författare)
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Structural and Functional Studies of Diiron Carboxylate Proteins
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Iron is essential to all life; it is a vital component of many proteins in humans as well as plants and bacteria. Because of the ability of iron to activate oxygen, it is often found in proteins that interact with oxygen in some way. One of the protein families that utilize iron to harness the oxidative power of the oxygen molecule for different purposes is the diiron carboxylate protein family. These proteins have two iron ions bound in the active site that are coordinated by four carboxylic residues and two histidines. These enzymes catalyse reactions such as radical generation, ubiquinol oxidation, fatty acid desaturation, iron oxidation and many different hydroxylation reactions. One subgroup of this protein family that recently has been discovered contains the membrane-bound diiron carboxylate proteins. In this thesis we have studied three diiron carboxylate proteins. The first, alternative oxidase, is a membrane-bound ubiquinol oxidase present in the respiratory chain of plants, some yeasts and protozoa. We have obtained the first spectroscopical evidence for the presence of a diiron carboxylate site in alternative oxidase. We have also identified the first prokaryotic alternative oxidase and shown it to be expressed and functional. The second membrane-bound diiron carboxylate protein studied is Coq7; the inactivation of this protein has been reported to prolong the life span of the model organism Caenorhabditis elegans. We have shown that Coq7 catalyses a hydroxylation in the biosynthesis of ubiquinone and identified it as a diiron carboxylate protein. Finally we have solved the structure of the ribonucleotide reductase R2 subunit from Chlamydia trachomatis, a soluble diiron carboxylate protein. We have discovered that this is an unusual member of the R2 family because it challenges the generally accepted dogma of a conserved radical-harbouring tyrosine being a requirement for enzyme activity in this protein class. We have by EPR spectroscopy demonstrated that the radical in this protein is stored as a high-valent species at the diiron site instead of on a tyrosine. We also found that many organisms, including several human pathogens, have R2 proteins with the same sequence abnormalities and propose that these proteins constitute a new ribonucleotide reductase subclass, class Ic.
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| 6. |
- Öhrström, Maria, et al.
(författare)
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Inhibition of chlamydial class Ic ribonucleotide reductase by C-terminal peptides from protein R2
- 2011
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 17:11, s. 756-762
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese-iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C-terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical-containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese-iron cofactor is similar to that of the tyrosyl-radical-containing RNRs, involving reversible long-range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2-derived oligopeptide blocks the transfer pathway. We have constructed three-dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20-mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C-terminus of protein R2.
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