21 results on '"Jianghui Meng"'
Search Results
2. Urotensin II/GPR14 Pathway Regulates Chronic Itch in Mice
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Hua Yang, Renkai Zhu, Wenhao Zhang, Weiwei Chen, Xinrong Yan, Chunxu Shan, Shanghai Xue, Ruizhen Wang, Xiaolong Dai, Jinhai Wang, Ciara Larkin, Jiafu Wang, and Jianghui Meng
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Cell Biology ,Dermatology ,Molecular Biology ,Biochemistry - Published
- 2023
3. Intervening GSK3 Signaling Attenuates Cutaneous Inflammation and Itch in Mice: Implication for Future Therapeutic Development
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Renkai Zhu, Hua Yang, Shanghai Xue, Qianqian Fan, Wenhao Zhang, Ruizhen Wang, Weiwei Chen, Lianlian Li, Jiafu Wang, and Jianghui Meng
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Cell Biology ,Dermatology ,Molecular Biology ,Biochemistry - Published
- 2023
4. Inhibiting Keratinocyte-Derived Signal Transducer and Activator of Transcription 6 Improved Atopic Dermatitis in Mice
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Shanghai Xue, Renkai Zhu, Weiwei Chen, Hua Yang, Jiafu Wang, and Jianghui Meng
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Cell Biology ,Dermatology ,Molecular Biology ,Biochemistry - Published
- 2022
5. Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release
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Jiafu Wang, Miaomiao Kong, Xiaolong Dai, Yu Dou, Yang Liu, Weiwei Chen, Shanghai Xue, Yanqing Li, Wanzhi Wang, Yinghao Zhang, and Jianghui Meng
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Male ,Membrane Fusion ,Rats, Sprague-Dawley ,Transient receptor potential channel ,Mice ,Pirt ,TAC1 ,cytokine ,Neurotoxin ,Botulinum Toxins, Type A ,Biology (General) ,Receptor ,Spectroscopy ,G alpha subunit ,Chemistry ,Nociceptors ,dorsal root ganglia ,General Medicine ,gene therapy ,Computer Science Applications ,Cell biology ,SNARE ,Female ,chronic pain ,Neuropeptide Y receptor Y2 ,Sensory Receptor Cells ,Synaptosomal-Associated Protein 25 ,QH301-705.5 ,Transgene ,Neuropeptide ,Pain ,neuronal promoter ,Catalysis ,Article ,Inorganic Chemistry ,Peripheral Nervous System ,Animals ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,neuropeptide ,QD1-999 ,Organic Chemistry ,Neuropeptides ,Membrane Proteins ,neurotoxin ,Rats ,Mice, Inbred C57BL ,Animals, Newborn - Abstract
Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1,  , CALCB, calcitonin gene-related peptide 2, HTR3A, 5-hydroxytryptamine receptor 3A, NPY2R, neuropeptide Y receptor Y2, GPR52, G protein-coupled receptor 52, SCN9A, sodium voltage-gated channel alpha subunit 9, TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.
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- 2021
6. A SNAP-25 cleaving chimera of botulinum neurotoxin /A and /E prevents TNFα−induced elevation of the activities of native TRP channels on early postnatal rat dorsal root ganglion neurons
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J. Oliver Dolly, Jianghui Meng, Yamil R. Yusef, Jiafu Wang, and Marc Nugent
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0301 basic medicine ,Botulinum Toxins ,Sensory Receptor Cells ,Synaptosomal-Associated Protein 25 ,TRPV1 ,TRPV Cation Channels ,Rats, Sprague-Dawley ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Transient receptor potential channel ,Dorsal root ganglion ,Downregulation and upregulation ,Ganglia, Spinal ,Escherichia coli ,medicine ,Animals ,Ankyrin ,Botulinum Toxins, Type A ,Cells, Cultured ,Pharmacology ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Tumor Necrosis Factor-alpha ,Fusion protein ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Animals, Newborn ,chemistry ,Capsaicin ,Sensory System Agents ,Neuropathic pain ,Calcium - Abstract
Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we firstly demonstrate by immuno-cytochemical labelling that TNFα augments the surface content of these channels on rat cultured dorsal root ganglion (DRG) neurons which, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNFα-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) and reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNFα-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNFα-dependent augmented, but not resting, surface trafficking of TRPV1/A1. As TNFα is known to induce nociceptive hyper-sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.
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- 2018
7. Role of SNAREs in Atopic Dermatitis-Related Cytokine Secretion and Skin-Nerve Communication
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Joerg Buddenkotte, Martin Steinhoff, Timo Buhl, Jiafu Wang, and Jianghui Meng
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0301 basic medicine ,Keratinocytes ,Stromal cell ,Thymic stromal lymphopoietin ,Sensory Receptor Cells ,Vesicle-Associated Membrane Protein 3 ,medicine.medical_treatment ,Inflammation ,Dermatology ,Biochemistry ,Dermatitis, Atopic ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Organ Culture Techniques ,Medicine ,Animals ,Humans ,Receptor ,Molecular Biology ,Cells, Cultured ,Toll-like receptor ,Endothelin-1 ,business.industry ,Cell Biology ,Immunity, Innate ,3. Good health ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Cytokine ,Ethylmaleimide ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques ,Immunology ,Cytokines ,Tumor necrosis factor alpha ,Cytokine secretion ,medicine.symptom ,Epidermis ,business ,SNARE Proteins - Abstract
The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in atopic dermatitis (AD) is unknown. This study identifies the function of soluble N-ethylmaleimide sensitive factor attachment protein receptor in AD-related cytokine secretion and epidermis-nerve communication. Herein, we report that various cytokines were simultaneously upregulated and coreleased in innate immunity–activated primary human keratinocytes. AD-related cytokines thymic stromal lymphopoietin, endothelin-1, and inflammatory tumor necrosis factor-α activated distinct but overlapping sensory neurons. Tumor necrosis factor-α potentiated thymic stromal lymphopoietin–induced Ca2+-influx, whereas endothelin-1 caused itch-selective B-type natriuretic peptide release. In primary human keratinocytes, B-type natriuretic peptide upregulated genes promoting dermatological and neuroinflammatory diseases and conditions. VAMP3, SNAP-29, and syntaxin 4 proved important in driving cytokine release from primary human keratinocytes. Depletion of VAMP3 inhibited nearly all the cytokine release including thymic stromal lymphopoietin and endothelin-1. Accordingly, VAMP3 co-occurred with endothelin-1 in the skins of patients with AD. Our study pinpoints the pivotal role of soluble N-ethylmaleimide sensitive factor attachment protein receptors in mediating cytokine secretion related to AD. VAMP3 is identified as a suitable target for developing broad-spectrum anticytokine therapeutics for controlling itch and atopic skin inflammation.
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- 2019
8. TLR3 in Chronic Human Itch: A Keratinocyte-Associated Mechanism of Peripheral Itch Sensitization
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Ian McDonald, Attila Gábor Szöllősi, Ellen H. van den Bogaard, Jianghui Meng, Martin Steinhoff, and Szabó I
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Keratinocytes ,Dermatology ,Biochemistry ,Mice ,chemistry.chemical_compound ,Thymic Stromal Lymphopoietin ,Psoriasis ,medicine ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,Sensitization ,Skin ,Toll-like receptor ,Endothelin-1 ,Interleukin-6 ,business.industry ,Pruritus ,Cell Biology ,Atopic dermatitis ,medicine.disease ,Toll-Like Receptor 3 ,Up-Regulation ,Poly I-C ,medicine.anatomical_structure ,chemistry ,Polyinosinic:polycytidylic acid ,Chronic Disease ,TLR3 ,Immunology ,Cytokines ,Tumor necrosis factor alpha ,Keratinocyte ,business - Published
- 2019
9. Role of SNARE proteins in tumourigenesis and their potential as targets for novel anti-cancer therapeutics
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Jiafu Wang and Jianghui Meng
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Cancer Research ,Carcinogenesis ,Kinase ,Autophagy ,Cancer ,Lipid bilayer fusion ,Antineoplastic Agents ,Oncogenes ,Biology ,medicine.disease ,Cell biology ,Paracrine signalling ,Oncology ,Neoplasms ,Cancer cell ,Genetics ,medicine ,Humans ,SNARE Proteins ,Autocrine signalling ,Calcium signaling - Abstract
The function of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in cellular trafficking, membrane fusion and vesicle release in synaptic nerve terminals is well characterised. Recent studies suggest that SNAREs are also important in the control of tumourigenesis through the regulation of multiple signalling and transportation pathways. The majority of published studies investigated the effects of knockdown/knockout or overexpression of particular SNAREs on the normal function of cells as well as their dysfunction in tumourigenesis promotion. SNAREs are involved in the regulation of cancer cell invasion, chemo-resistance, the transportation of autocrine and paracrine factors, autophagy, apoptosis and the phosphorylation of kinases essential for cancer cell biogenesis. This evidence highlights SNAREs as potential targets for novel cancer therapy. This is the first review to summarise the expression and role of SNAREs in cancer biology at the cellular level, their interaction with non-SNARE proteins and modulation of cellular signalling cascades. Finally, a strategy is proposed for developing novel anti-cancer therapeutics using targeted delivery of a SNARE-inactivating protease into malignant cells.
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- 2015
10. Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain
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J. Oliver Dolly, Jianghui Meng, Marc Nugent, Minhong Tang, and Jiafu Wang
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0301 basic medicine ,Sensory Receptor Cells ,media_common.quotation_subject ,Primary Cell Culture ,Mutant ,Synaptic vesicle ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,medicine ,Animals ,Humans ,Paralysis ,Botulinum Toxins, Type A ,Internalization ,media_common ,Neurons ,Membrane Glycoproteins ,Multidisciplinary ,Chemistry ,Cell Membrane ,Neurotoxicity ,Wild type ,medicine.disease ,Acetylcholine ,Recombinant Proteins ,Rats ,3. Good health ,Cell biology ,030104 developmental biology ,Membrane protein ,Cell culture ,Mutant Proteins ,Synaptic Vesicles ,030217 neurology & neurosurgery ,Protein Binding ,medicine.drug - Abstract
Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 104-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with 125I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons.
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- 2017
11. Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons
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J. Oliver Dolly, Jiafu Wang, Gary W. Lawrence, and Jianghui Meng
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Dynamins ,Botulinum Toxins ,Mice, 129 Strain ,media_common.quotation_subject ,Neurotoxins ,Adaptor Protein Complex 2 ,Glutamic Acid ,Mice, Transgenic ,Nerve Tissue Proteins ,Endocytosis ,Biochemistry ,Synaptic vesicle ,Clathrin ,Bulk endocytosis ,Rats, Sprague-Dawley ,Mice ,Adaptor Protein Complex alpha Subunits ,Genetics ,Animals ,Protein Isoforms ,Botulinum Toxins, Type A ,Internalization ,Molecular Biology ,Cells, Cultured ,Dynamin ,media_common ,Mice, Knockout ,Neurons ,Microscopy, Confocal ,biology ,Secretory Vesicles ,Signal transducing adaptor protein ,Rats ,Cell biology ,Mice, Inbred C57BL ,Mutation ,Amphiphysin ,biology.protein ,RNA Interference ,Synaptic Vesicles ,Peptides ,Biotechnology - Abstract
Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.
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- 2013
12. Distinct functions of dynamin isoforms in tumorigenesis and their potential as therapeutic targets in cancer
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Jianghui Meng
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0301 basic medicine ,Dynamins ,endocrine system ,tumor ,Carcinogenesis ,macromolecular substances ,Review ,medicine.disease_cause ,Endocytosis ,Clathrin ,Metastasis ,03 medical and health sciences ,clathrin ,Neoplasms ,medicine ,Humans ,Protein Isoforms ,endocytosis ,amphiphysin ,Dynamin ,biology ,Cancer ,Cell migration ,Biological Transport ,medicine.disease ,Cell biology ,030104 developmental biology ,Oncology ,Amphiphysin ,biology.protein ,cancer target ,biological phenomena, cell phenomena, and immunity - Abstract
// Jianghui Meng 1,2 1 Charles Institute of Dermatology, School of Medicine and Medical Sciences, University College Dublin, Belfield, Dublin, Ireland 2 International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin, Ireland Correspondence to: Jianghui Meng, email: // email: // Keywords : endocytosis, tumor, cancer target, clathrin, amphiphysin Received : September 28, 2016 Accepted : March 09, 2017 Published : March 29, 2017 Abstract Dynamins and their related proteins participate in the regulation of neurotransmission, antigen presentation, receptor internalization, growth factor signalling, nutrient uptake, and pathogen infection. Recently, emerging findings have shown dynamin proteins can also contribute to the genesis of cancer. This up-to-date review herein focuses on the functionality of dynamin in cancer development. Dynamin 1 and 2 both enhance cancer cell proliferation, tumor invasion and metastasis, whereas dynamin 3 has tumor suppression role. Antisense RNAs encoded on the DNA strand opposite a dynamin gene regulate the function of dynamin, and manipulate oncogenes and tumor suppressor genes. Certain dynamin-related proteins are also upregulated in distinct cancer conditions, resulting in apoptotic resistance, cell migration and poor prognosis. Altogether, dynamins are potential biomarkers as well as representing promising novel therapeutic targets for cancer treatment. This study also summarizes the current available dynamin-targeted therapeutics and suggests the potential strategy based on signalling pathways involved, providing important information to aid the future development of novel cancer therapeutics by targeting these dynamin family members.
- Published
- 2016
13. TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18–1/syntaxin1/SNAP-25 mediated fusion
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Jiafu Wang, Martin Steinhoff, James Oliver Dolly, and Jianghui Meng
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0301 basic medicine ,Sensory Receptor Cells ,Synaptosomal-Associated Protein 25 ,Vesicle-Associated Membrane Protein 1 ,Syntaxin 1 ,TRPV Cation Channels ,Calcitonin gene-related peptide ,Synaptic vesicle ,Membrane Fusion ,Exocytosis ,Article ,Rats, Sprague-Dawley ,03 medical and health sciences ,Transient receptor potential channel ,0302 clinical medicine ,Munc18 Proteins ,Syntaxin ,Animals ,Humans ,Calcium Signaling ,TRPA1 Cation Channel ,TRPC Cation Channels ,Multidisciplinary ,Chemistry ,Synaptic vesicle membrane ,Tumor Necrosis Factor-alpha ,Vesicle ,Molecular biology ,Transport protein ,Cell biology ,Rats ,Protein Transport ,030104 developmental biology ,nervous system ,Synaptic Vesicles ,030217 neurology & neurosurgery - Abstract
Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca2+ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.
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- 2016
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14. Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis
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Jianghui Meng, J. Oliver Dolly, Jiafu Wang, Gary W. Lawrence, K. Roger Aoki, Larry A. Wheeler, and Tomas H. Zurawski
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Proteases ,Botulinum Toxins ,Synaptosomal-Associated Protein 25 ,Recombinant Fusion Proteins ,Blotting, Western ,Neurotoxins ,Neurotransmission ,Biology ,medicine.disease_cause ,Biochemistry ,Neuromuscular junction ,Rats, Sprague-Dawley ,Mice ,Chimera (genetics) ,Tetanus Toxin ,Cerebellum ,Genetics ,medicine ,Animals ,Paralysis ,Receptor ,Molecular Biology ,Neurons ,Toxin ,Neuromuscular Diseases ,Sciatic Nerve ,Virology ,In vitro ,Rats ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Spinal Cord ,Mutation ,Axoplasmic transport ,Peptide Hydrolases ,Biotechnology - Abstract
Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.
- Published
- 2012
15. Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B
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J. Oliver Dolly, MacDara Bodeker, Tomas H. Zurawski, Jiafu Wang, Sanjay V. Boddul, K. Roger Aoki, and Jianghui Meng
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Botulinum Toxins ,Synaptosomal-Associated Protein 25 ,Vesicle-Associated Membrane Protein 2 ,Recombinant Fusion Proteins ,medicine.medical_treatment ,Urinary Bladder ,Neuromuscular Junction ,In Vitro Techniques ,Immunoglobulin light chain ,Synaptic Transmission ,Biochemistry ,Synaptic vesicle ,Exocytosis ,Synaptotagmin 1 ,Green fluorescent protein ,Rats, Sprague-Dawley ,Mice ,medicine ,Animals ,Paralysis ,Botulinum Toxins, Type A ,Receptor ,Molecular Biology ,Cells, Cultured ,Neurons ,Protease ,Chemistry ,Synovial Membrane ,Muscle, Smooth ,Cell Biology ,Fibroblasts ,Molecular biology ,Protein Structure, Tertiary ,Rats ,Phrenic Nerve ,Membrane protein ,Female ,Peptide Hydrolases - Abstract
Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [HC/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting HC/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis. Abbreviations: BoNT, botulinum neurotoxin; CGN, cerebellar granule neuron; DAS, digit abduction score; DC, di-chain; DTT, dithiothreitol; GFP, green fluorescent protein; GST, glutathione transferase; HC, heavy chain; HC, C-terminal half of HC; HN, N-terminal half of HC; IMAC, immobilized metal-affinity chromatography; LC, light chain; mLD50, mouse LD50; SC, single chain; SNAP-23, synaptosome-associated protein of 23 kDa; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SV2, synaptic vesicle protein 2; Syt, synaptotagmin; TDmax., maximal tolerated dose; VAMP, vesicle-associated membrane protein
- Published
- 2012
16. Novel therapeutics based on recombinant botulinum neurotoxins to normalize the release of transmitters and pain mediators
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J. Oliver Dolly, Jianghui Meng, Tomas H. Zurawski, and Jiafu Wang
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chemistry.chemical_classification ,Proteases ,Protease ,medicine.medical_treatment ,Chronic pain ,Cell Biology ,Pharmacology ,medicine.disease ,Biochemistry ,Exocytosis ,law.invention ,Transient receptor potential channel ,Enzyme ,Nociception ,chemistry ,law ,medicine ,Recombinant DNA ,Molecular Biology - Abstract
A major unmet clinical need exists for long-acting neurotherapeutics to alleviate chronic pain in patients unresponsive to available nonaddictive analgesics. Herein, a new strategy is described for the development of potent and specific inhibitors of the neuronal exocytosis of transmitters and pain mediators that exhibit unique antinociceptive activity. This entailed recombinant production in Escherichia coli of two serotypes of botulinum neurotoxin (BoNT) (BoNT(A) and BoNT(E) ), which are proteins that are known to block the release of transmitters by targeting and entering nerve endings, where their proteases cleave and inactivate a protein, synaptosomal protein of M(r) 25 000 (SNAP-25), that is essential for Ca(2+) -regulated exocytosis. Site-directed mutagenesis of Leu428 and Leu429 in BoNT(A) revealed that the remarkable longevity of its neuroparalytic action is attributable to a dileucine-containing motif. BoNT(E) acts transiently, because it lacks these residues, but is a superior inhibitor of transient receptor potential vanilloid type 1-mediated release of pain peptides from sensory nerves. The advantageous features of each serotype were harnessed by attaching the BoNT(E) protease moiety to an enzymically inactive mutant of BoNT(A) . The resultant purified composite protein could target motoneurons by binding to the BoNT(A) ectoacceptor and persistently produce BoNT(E) -truncated SNAP-25. As this enzyme lasted for more than 1 month (as compared with 5 days for BoNT(E) alone), such a dramatic extension in the lifetime of this BoNT(E) protease is attributable to a stabilizing influence of the BoNT(A) mutant. Most importantly, injecting this novel biotherapeutic into the foot pads of rats resulted in extended amelioration of inflammatory pain. Thus, a new generation of biotherapeutics has been created with the potential to give long-term relief of pain.
- Published
- 2011
17. A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis
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Jiafu Wang, Weredeselam M. Olango, Tomas H. Zurawski, Gary W. Lawrence, Jianghui Meng, David P. Finn, Larry A. Wheeler, and J. Oliver Dolly
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medicine.medical_specialty ,neuromuscular-transmission ,Neuromuscular transmission ,translocation ,Biology ,Pharmacology ,Inhibitory postsynaptic potential ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Transient receptor potential channel ,Internal medicine ,medicine ,Botulism ,Neurotransmitter ,Molecular Biology ,Neurotoxicity ,persistence ,Cell Biology ,medicine.disease ,Botulinum toxin ,inhibition ,synaptotagmin-i ,sensory neurons ,Endocrinology ,injection ,chemistry ,Clostridium botulinum ,nerve-terminals ,exocytosis ,neurotoxin-e ,medicine.drug - Abstract
Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E)-BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.
- Published
- 2011
18. Synaptobrevin I mediates exocytosis of CGRP from sensory neurons and inhibition by botulinum toxins reflects their anti-nociceptive potential
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J. Oliver Dolly, Jianghui Meng, Jiafu Wang, and Gary W. Lawrence
- Subjects
Botulinum Toxins ,Synaptosomal-Associated Protein 25 ,Synaptobrevin ,Calcitonin Gene-Related Peptide ,TRPV1 ,Pain ,Syntaxin 1 ,Substance P ,Calcitonin gene-related peptide ,Biology ,Bradykinin ,Synaptic vesicle ,Exocytosis ,Synaptotagmin 1 ,Membrane Potentials ,R-SNARE Proteins ,Mice ,Animals ,Protein Isoforms ,Neurons, Afferent ,Nociceptors ,SNAP25 ,Cell Biology ,Rats ,Cell biology ,Trigeminal Ganglion ,nervous system ,Biochemistry ,Potassium ,Calcium ,Synaptic Vesicles ,Capsaicin ,SNARE Proteins - Abstract
Calcitonin-gene-related peptide (CGRP), a potent vasodilator that mediates inflammatory pain, is elevated in migraine; nevertheless, little is known about its release from sensory neurons. In this study, CGRP was found to occur in the majority of neurons from rat trigeminal ganglia, together with the three exocytotic SNAREs [SNAP25, syntaxin 1 and the synaptobrevin (Sbr, also known as VAMP) isoforms] and synaptotagmin. Ca2+-dependent CGRP release was evoked with K+-depolarisation and, to lower levels, by capsaicin or bradykinin from neurons that contain the vanilloid receptor 1 and/or bradykinin receptor 2. Botulinum neurotoxin (BoNT) type A cleaved SNAP25 and inhibited release triggered by K+ > bradykinin >> capsaicin. Unlike BoNT type D, BoNT type B did not affect exocytosis, even though the neurons possess its receptor and Sbr II and Sbr III got proteolysed (I is resistant in rat) but, in mouse neurons, it additionally cleaved Sbr I and blocked transmitter release. Sbr I and II were found in CGRP-containing vesicles, and each was shown to separately form a SNARE complex. These new findings, together with punctate staining of Sbr I and CGRP in neurites, implicate isoform Sbr I in exocytosis from large dense-core vesicles together with SNAP25 (also, probably, syntaxin 1 because BoNT type C1 caused partial cleavage and inhibition); this differs from Sbr-II-dependent release of transmitters from small synaptic vesicles. Such use of particular Sbr isoform(s) by different neurons raises the functional implications for other cells previously unrecognised.
- Published
- 2007
19. Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X(3) inhibits the release of a pain mediator
- Author
-
J. Oliver Dolly, Stephen Hearty, Hui Ma, Jianghui Meng, Richard O'Kennedy, and Jiafu Wang
- Subjects
Phage display ,Sensory Receptor Cells ,Synaptosomal-Associated Protein 25 ,medicine.medical_treatment ,Calcitonin Gene-Related Peptide ,Recombinant Fusion Proteins ,Pain ,Peptide ,Calcitonin gene-related peptide ,Biochemistry ,Mice ,Dorsal root ganglion ,Ganglia, Spinal ,medicine ,Extracellular ,Animals ,Humans ,Botulinum Toxins, Type A ,Molecular Biology ,Cells, Cultured ,chemistry.chemical_classification ,Protease ,Chemistry ,Cell Biology ,Fusion protein ,Molecular biology ,Rats ,medicine.anatomical_structure ,Neuropathic pain ,Female ,Rabbits ,Receptors, Purinergic P2X3 ,Single-Chain Antibodies - Abstract
P2X3 (P2X purinoceptor 3) is predominantly expressed on nociceptive sensory neurons and plays a crucial role in signalling leading to chronic inflammatory pain and some features of neuropathic pain. Thus it represents a potential target for pain therapeutics. BoNT/A (botulinum neurooxin type A) effectively relieves certain types of pain through inhibiting the neuronal release of pain peptides. A recombinant single-chain variable fragment (scFv) antibody designated MH7C was generated against the extracellular domain of P2X3 using phage display. The genes encoding the scFv and activated di-chain form of BoNT/A without the C-terminal-binding subdomain (LC–HN–HCN/A) were ligated and expressed in Escherichia coli cells as a composite fusion protein. The purified protein bound and entered P2X3-containing sensory neurons, cleaved synaptosomal-associated protein of 25 kDa and inhibited the release of a pain peptide. This novel fusion protein designated ‘LC–HN–HCN/A–MH7C’ has potential clinical applications in the treatment of chronic inflammatory and sympathetically maintained neuropathic pain. Abbreviations: A.U., arbitrary unit; BoNT, botulinum neurotoxin; CGN, cerebellar granule neuron; CGRP, calcitonin gene-related peptide; DIV, days in vitro; DPBS, Dulbecco’s PBS; DRG, dorsal root ganglion; HA, haemagglutinin; HRP, horseradish peroxidase; IMAC, immobilized metal-ion-affinity chromatography; LC–HN–HCN/A, protease light chain, translocation and N-terminal-binding subdomain of BoNT/A; LC, light chain of BoNT; β-MCD, methyl-β-cyclodextrin; P, postnatal day; PBST, PBS with 1:1000 (v/v) Tween 20; PM, plasma membrane; P2X, P2X purinoceptor; scFv, single-chain Fv fragment; SNAP-25, synaptosome-associated protein of 25 kDa
- Published
- 2014
20. SNARE complexes mediate inflammation-induced co-trafficking of TRPV1/TRPA1 to the surface of sensory neurons and neuropeptide exocytosis: Inhibition by botulinum neurotoxins highlights their antinociceptive potential
- Author
-
Jiafu Wang, Jianghui Meng, James Oliver Dolly, and Martin Steinhoff
- Subjects
Nociception ,Chemistry ,Exocytosis Inhibition ,medicine ,TRPV1 ,Neuropeptide ,Inflammation ,Sensory system ,medicine.symptom ,Toxicology ,Cell biology - Published
- 2016
21. Role of snare proteins in tumorigenesis and protein engineering: SNARE-inactivating anti-cancer therapeutics
- Author
-
Jianghui Meng and Jiafu Wang
- Subjects
medicine ,Cancer ,Protein engineering ,Biology ,Toxicology ,Carcinogenesis ,medicine.disease_cause ,medicine.disease ,Cell biology - Published
- 2016
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