Biology subjects

Beeson, D.

Publications and source records attributed to Beeson, D..

5 recordsLinked to original sources

bioRxiv2024

Structures of the human adult muscle-type nicotinic receptor in resting and desensitised states

Muscle-type nicotinic acetylcholine receptor (AChR) is the key signalling molecule in neuromuscular junctions. Here we present the structures of full-length human adult receptors in complex with Fab35 in -bungarotoxin (BuTx)-bound resting states, and in acetylcholine (ACh)-bound desensitised states. In addition to identifying the conformational changes during recovery from desensitisation, we also used electrophysiology to probe the effects of eight previously unstudied AChR genetic variants found in congenital myasthenic syndrome (CMS) patients, revealing they cause either slow- or fast-channel CMS characterised by prolonged or abbreviated ion channel bursts. The combined kinetic and structural data offer a better understanding of both AChR state transition and the pathogenic mechanisms of disease variants.

neuroscience↗

bioRxiv2024

Dose escalation pre-clinical trial of novel Dok7-AAV in mouse model of DOK7 congenital myasthenia

Congenital myasthenic syndromes (CMS) are a group of inherited disorders characterised by defective neuromuscular transmission and fatigable muscle weakness. Mutations in DOK7, a gene encoding a post-synaptic protein crucial in the formation and stabilisation of the neuromuscular junction (NMJ), rank among the leading three prevalent causes of CMS in diverse populations globally. The majority of DOK7 CMS patients experience varying degrees of disability despite receiving optimised treatment, necessitating the development of improved therapeutic approaches. Here we executed a dose escalation pre-clinical trial using a DOK7-CMS mouse model to assess the efficacy of Amp-101, an innovative AAV gene replacement therapy. Amp-101 is based on AAVrh74 and contains human DOK7 cDNA under the control of a muscle-restricted promoter. We show that at doses 6x1013vg/kg and 1x1014 vg/kg, Amp-101 generated enlarged NMJs and rescued the very severe phenotype of the model. Treated mice became at least as strong as WT littermates and the diaphragm and tibialis anterior muscles displayed robust expression of DOK7. This data suggests that Amp-101 is a promising candidate to move forward to clinic trials.

neuroscience↗

bioRxiv2020

Structural correlates of human muscle nicotinic acetylcholine receptor subunit assembly mediated by δ(+) interface residues

Muscle nicotinic acetylcholine receptors are a class of heteropentameric ligand-gated cation channels with constituent subunits adopting a fixed stoichiometric arrangement. The specific amino acid residues that govern subunit ordering are however, only partially understood. By integrating all-atom molecular dynamics simulations, bioinformatics, two-electrode voltage clamp electrophysiology and 125I--bungarotoxin assays of chimeric nAChR subunits, we identify residues across the extracellular, transmembrane and extended M4 helix of the {delta} subunit that make structural signatures that contribute to intransigent assembly rules. Furthermore, functional differences observed in 2{delta}2{beta} receptors can be rationalized by changes in dynamical behavior that manifest themselves at the agonist binding site.

neuroscience↗

bioRxiv2018

Isolation and characterization of rare circulating autoantibody-producing cells from patients with muscle-specific kinase myasthenia gravis

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by autoantibodies that bind to functional membrane proteins at the neuromuscular junction. Most patients have autoantibodies to the acetylcholine receptor (AChR), but a subset of patients instead have autoantibodies to muscle specific tyrosine kinase (MuSK). MuSK is an essential component of the Agrin/Lipoprotein receptor-related protein 4(LRP4)/MuSK/downstream of tyrosine kinase 7 (DOK7) pathway that is responsible for synaptic differentiation, including clustering of AChRs at the neuromuscular junction, both during development and in adult muscle. Nerve-released Agrin binds to LRP4 which then binds to MuSK, stimulating autophosphorylation and recruitment of DOK7 to complete the membrane component of the pathway. Serum-derived IgG4 subclass MuSK autoantibodies prevent the binding of LRP4 to MuSK, subsequently impairing autophosphorylation, resulting in the loss of Agrin-induced AChR clustering in the mouse myotube-forming C2C12 line. Although this autoimmune mechanism appears well understood, MuSK autoantibodies from patients are polyclonal. Most are IgG4 but IgG1, 2 and 3 are also present and can achieve similar results on AChR clustering. In addition, most bind the first Ig-like domain in MuSK, however some patients harbor serum autoantibodies that recognize other domains in MuSK. We sought to establish individual MuSK IgG clones so that the disease mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from MuSK MG patients with a fluorescent-tagged MuSK antigen multimer and generated a panel of human monoclonal autoantibodies (mAbs) from these cells. We produced 77 mAbs from single B cells collected from six MuSK MG patients. Here we focused on three highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells and at mouse neuromuscular junctions where they co-localized with AChRs. These three IgG isotype mAbs (two IgG4 and one IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The three MuSK mAbs inhibited Agrin induced-AChR clustering in C2C12 myotubes, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation. This approach for ex vivo isolation of MuSK MG autoantibody-producing cells and production of human recombinant mAbs has identified distinct autoantibody specificities and likely divergent effector mechanisms. Collectively, these findings will enable a better understanding of MuSK autoantibody-mediated pathology.

immunology↗

bioRxiv2018

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic lipid-altered tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.\n\nHighlightsO_LIStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis\nC_LIO_LIDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity\nC_LIO_LIStructures, kinetics and biosynthesis reveal role of lipid in tunicamycin\nC_LIO_LILipid-altered, tunicamycin analogues give non-toxic antibiotics against TB\nC_LI

biochemistry↗