scholarly journals α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli

Marine Drugs ◽  
2020 ◽  
Vol 18 (8) ◽  
pp. 422
Author(s):  
Yanli Liu ◽  
Yifeng Yin ◽  
Yunyang Song ◽  
Kang Wang ◽  
Fanghui Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
High Performance ◽  
Cognitive Disorders ◽  
Coli Strain ◽  
Xenopus Laevis Oocytes ◽  
Obvious Effect ◽  
Nicotinic Acetylcholine ◽  
Α7 Nachr

α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer’s and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.

scholarly journals Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 603 ◽  
Author(s):  
Jiong Ning ◽  
Jie Ren ◽  
Yang Xiong ◽  
Yong Wu ◽  
Manqi Zhangsun ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Fold Increase ◽  
Cd Spectroscopy ◽  
Xenopus Laevis Oocytes ◽  
Muscle Type ◽  
Nicotinic Acetylcholine ◽  
Activity Loss ◽  
Spectroscopy Studies ◽  
Key Residues

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure–activity relationship (SAR) of EI revealed that the “Arg1–Asn2–Hyp3” residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1–3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1–3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

Molecular cloning and characterization of a novel human variant of RIC-3, a putative chaperone of nicotinic acetylcholine receptors

2008 ◽  
Vol 28 (6) ◽  
pp. 299-306 ◽  
Author(s):  
Tamara Seredenina ◽  
Teresa Ferraro ◽  
Georg C. Terstappen ◽  
Andrea Caricasole ◽  
Renza Roncarati
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Coiled Coil ◽  
Surface Expression ◽  
Protein Product ◽  
Nicotinic Acetylcholine ◽  
Α7 Nachr ◽  
New Variant ◽  
Novel Variant

Recent reports demonstrate that the RIC-3 (resistant to inhibitors of cholinesterase-3) protein is important for the maturation of nAChRs (nicotinic acetylcholine receptors). In the present study RIC-3e, a novel variant of RIC-3, is described. This variant contains a deletion of exons 4 and 5 of RIC-3, resulting in a protein product lacking a conserved coiled-coil domain. Like RIC-3, the new variant is predominantly, but not exclusively, expressed in the brain. The analysis of expression of variant RIC-3 mRNA and of α7-nAChR mRNA in a set of human tissues shows a similar profile. The RIC-3e protein is functionally active and enables surface expression of mature α7-nAChRs in cell lines not otherwise permissive for the expression of this receptor.

scholarly journals Nicotinic acetylcholine receptor partial antagonist polyamides from tunicates and their predatory sea slugs

2021 ◽  
Author(s):  
Noemi D. Paguigan ◽  
Jortan O. Tun ◽  
Lee S. Leavitt ◽  
Zhenjian Lin ◽  
Kevin Chase ◽  
...  
Keyword(s):  
Nervous System ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Partial Agonist ◽  
Muscarinic Acetylcholine Receptors ◽  
New Drugs ◽  
Calcium Flux ◽  
Nicotinic Acetylcholine ◽  
Chemical Structures ◽  
Α7 Nachr

In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii and their prey, Didemnum molle tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3β4 (mouse) and α6/α3β4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 μM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

Nicotinic Acetylcholine Receptors

2018 ◽  
Author(s):  
Roger L. Papke
Keyword(s):  
Ion Channels ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Cognitive Disorders ◽  
The Body ◽  
Nicotinic Acetylcholine ◽  
Synaptic Receptors ◽  
The Central Nervous System ◽  
The Brain

Acetylcholine, exquisitely evolved as a neurotransmitter, is made and released by the neurons that take the integrated output of the central nervous system throughout the body. At both neuromuscular junctions and autonomic ganglia, acetylcholine activates synaptic ion channels that take their name from the plant alkaloid nicotine, which is a mimic of the natural neurotransmitter. This chapter begins with the scientific discoveries related to the nicotinic acetylcholine receptors (nAChR) of the neuromuscular junction and how resulting insights led to an understanding of the fundamentals of synaptic transmission. The nAChR are one member of a superfamily of ligand-gated ion channels, and although in the brain excitatory neurotransmission is mediated by another family of synaptic receptors that are gated by glutamate, nicotinic receptors are important modulators of brain function and significant targets for drug development. In the brain, nAChR are targets for cognitive disorders and, tragically, responsible for tobacco addiction.

scholarly journals Migration of gastric cancer is suppressed by recombinant Newcastle disease virus (rL-RVG) via regulating α7-nicotinic acetylcholine receptors/ERK- EMT

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xuefeng Bu ◽  
Anwei Zhang ◽  
Zhengwei Chen ◽  
Xuanfeng Zhang ◽  
Riting Zhang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Nicotinic Acetylcholine Receptors ◽  
Newcastle Disease ◽  
Acetylcholine Receptors ◽  
Gastric Cancer Cells ◽  
Nicotinic Acetylcholine ◽  
Mesenchymal Transition ◽  
Α7 Nachr

Abstract Background Nicotinic acetylcholine receptors (nAChRs) have been reported to be overexpressed in malignancies in humans and is associated with tumorigenesis and cell migration. In previous studies of gastric cancer, alpha7 nicotinic acetylcholine receptor (α7-nAChR) overexpression leads to epithelial-mesenchymal transition (EMT) and promotes the migration of gastric cancer cells. Recombinant avirulent LaSota strain of Newcastle disease virus (NDV) expressing the rabies virus glycoprotein (rL-RVG) may promote apoptosis of gastric cancer cells and reduces the migration of lung cancer metastasis. However, whether rL-RVG inhibits migration of gastric cancer cells and what the underlying functional mechanism is remains unknown. Methods The gastric cancer cell lines BGC and SGC were randomly divided into 3 groups: rL-RVG, NDV and Phosphate Buffered Solution (PBS) control groups. Furthermore,we adopted ACB and MLA,α7nAChR-siRNA for the overexpression and silencing of α7-nAChR.Corynoxenine was used for inhibiting the MEK-ERK pathway. Western blot, Immunofluoresce,cell proliferation assays,cell migration analyses through wound-healing assays and Transwell assays were used to explore the underlying mechanisms. A mouse xenograft model was used to investigate the effects of rL-RVG,NDV on tumor growth. Results In this study, our findings demonstrate that rL-RVG suppressed the migration of gastric cancer cells and reduced EMT via α7-nAChR in vitro. Furthermore rL-RVG decreased the phosphorylation levels of the MEK/ERK signaling pathway such as down-regulating the expression of P-MEK and P-ERK. Additionally, rL-RVG also reduced the expression level of mesenchymal markers N-cadherin and Vimentin and enhanced the expression of the epithelial marker E-cadherin. Lastly, rL-RVG inhibited nicotinic acetylcholine receptors (nAChRs) to suppress cell migration and epithelial to mesenchymal transition (EMT) in gastric cell. We also found that rL-RVG suppresses the growth of gastric cancer subcutaneous tumor cells in vivo. Conclusion rL-RVG inhibits α7-nAChR-MEK/ERK-EMT to suppress migration of gastric cancer cells.

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