scholarly journals Two Novel Peptide Toxins from the Spider Cyriopagopus longipes Inhibit Tetrodotoxin-Sensitive Sodium Channels

Toxins ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 529
Author(s):  
Qingfeng Zhang ◽  
Yuxin Si ◽  
Li Yang ◽  
Li Wang ◽  
Shuijiao Peng ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Action Potentials ◽  
Inhibitory Concentration ◽  
Pharmacological Study ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Channel Activation ◽  
Peptide Toxins ◽  
A Site ◽  
Animal Venoms

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and Cl6b) were isolated from the venom of the spider Cyriopagopus longipes and characterized. Cl6a and Cl6b were shown to be inhibitors of tetrodotoxin-sensitive (TTX-S), but not TTX-resistant, sodium channels. Among the TTX-S channels investigated, Cl6a and Cl6b showed the highest degree of inhibition against NaV1.7 (half-maximal inhibitory concentration (IC50) of 11.0 ± 2.5 nM and 18.8 ± 2.4 nM, respectively) in an irreversible manner that does not alter channel activation, inactivation, or repriming kinetics. Moreover, analysis of NaV1.7/NaV1.8 chimeric channels revealed that Cl6b is a site 4 neurotoxin. Site-directed mutagenesis analysis indicated that D816, V817, and E818 observably affected the efficacy of the Cl6b-NaV1.7 interaction, suggesting that these residues might directly affect the interaction of NaV1.7 with Cl6b. Taken together, these two novel peptide toxins act as potent and sustained NaV1.7 blockers and may have potential in the pharmacological study of sodium channels.

Phosphorylation of Ser136 is critical for potent bone sialoprotein-mediated nucleation of hydroxyapatite crystals

2010 ◽  
Vol 428 (3) ◽  
pp. 385-395 ◽  
Author(s):  
Gurpreet S. Baht ◽  
Jason O'Young ◽  
Antonia Borovina ◽  
Hong Chen ◽  
Coralee E. Tye ◽  
...  
Keyword(s):  
Critical Role ◽  
Bone Sialoprotein ◽  
Site Directed Mutagenesis ◽  
Crystal Face ◽  
Mineralized Tissues ◽  
Dynamics Simulations ◽  
A Site ◽  
Functional Analyses ◽  
Kinase Ck2

Acidic phosphoproteins of mineralized tissues such as bone and dentin are believed to play important roles in HA (hydroxyapatite) nucleation and growth. BSP (bone sialoprotein) is the most potent known nucleator of HA, an activity that is thought to be dependent on phosphorylation of the protein. The present study identifies the role phosphate groups play in mineral formation. Recombinant BSP and peptides corresponding to residues 1–100 and 133–205 of the rat sequence were phosphorylated with CK2 (protein kinase CK2). Phosphorylation increased the nucleating activity of BSP and BSP-(133–205), but not BSP-(1–100). MS analysis revealed that the major site phosphorylated within BSP-(133–205) was Ser136, a site adjacent to the series of contiguous glutamate residues previously implicated in HA nucleation. The critical role of phosphorylated Ser136 in HA nucleation was confirmed by site-directed mutagenesis and functional analyses. Furthermore, peptides corresponding to the 133–148 sequence of rat BSP were synthesized with or without a phosphate group on Ser136. As expected, the phosphopeptide was a more potent nucleator. The mechanism of nucleation was investigated using molecular-dynamics simulations analysing BSP-(133–148) interacting with the {100} crystal face of HA. Both phosphorylated and non-phosphorylated sequences adsorbed to HA in extended conformations with alternating residues in contact with and facing away from the crystal face. However, this alternating-residue pattern was more pronounced when Ser136 was phosphorylated. These studies demonstrate a critical role for Ser136 phosphorylation in BSP-mediated HA nucleation and identify a unique mode of interaction between the nucleating site of the protein and the {100} face of HA.

scholarly journals Critical Role of Cys168 in Noggin Protein's Biological Function

2005 ◽  
Vol 37 (3) ◽  
pp. 181-185
Author(s):  
Wei-Dong Liu ◽  
Xiang-Ling Feng ◽  
Cai-Ping Ren ◽  
Jian-Ling Shi ◽  
Xu-Yu Yang ◽  
...  
Keyword(s):  
Biological Activities ◽  
Critical Role ◽  
Ectopic Expression ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Morphogenetic Protein ◽  
Neural Cell Adhesion ◽  
A Site ◽  
The Relationship

Abstract Previous that noggin exerts its neural inducing effect by binding and antagonizing bone morphogenetic protein 4 (BMP4). In order to further clarify the relationship between the structure and the function of noggin, and elucidate the possible mechanism responsible for noggin-BMP4 interaction, we generated three noggin mutants, C168S, C174S and C197S, by using a site-directed mutagenesis method. Ectopic expression of wild-type (WT) noggin, C174S or C197S, in Xenopus animal caps (ACs) by mRNA injection converted the explants (prospective ectoderm) into neural tissue, as indicated by the neural-like morphology and expression of the neural cell adhesion molecule (NCAM) in the ACs. In contrast, ACs expressing C168S suffered an epidermal fate similar to the control caps. Similarly, among the three mutants, only C168S lost the dorsalizing function. These studies highlight the critical role played by Cys168 in noggin's biological activities. It probably participates in the formation of an intermolecular disulfide bridge.

scholarly journals Sodium channels implement a molecular leaky integrator that detects action potentials and regulates neuronal firing

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marco A Navarro ◽  
Autoosa Salari ◽  
Jenna L Lin ◽  
Luke M Cowan ◽  
Nicholas J Penington ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Action Potentials ◽  
Critical Role ◽  
Low Frequency ◽  
Kinetic Mechanism ◽  
Excitatory Input ◽  
Neuronal Firing ◽  
Na Current ◽  
Brain Functions ◽  
Leaky Integrator

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state. Through this mechanism, sodium channels effectively measure the frequency of action potentials and convert it into Na+ current availability. In turn, the Na+ current can control neuronal firing frequency in a negative feedback loop. Consequently, neurons become less sensitive to changes in excitatory input and maintain a lower firing rate. We present these ideas in the context of rat serotonergic raphe neurons, which fire spontaneously at low frequency and provide critical neuromodulation to many autonomous and cognitive brain functions.

Identification of a Novel Exosite (Glu634-Arg639) in the Spacer Domain of ADAMTS13 Required for Recognition of Von Willebrand Factor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2214-2214 ◽  
Author(s):  
Veronica Casina ◽  
Hayley Hanby ◽  
Anastasia Lyalenko ◽  
X. Long Zheng
Keyword(s):  
Proteolytic Activity ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
Substrate Recognition ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Evolutionarily Conserved ◽  
A Site ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2214 Exosite binding plays a key role in cleavage of VWF by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13). Two exosites that are evolutionarily conserved from zebra fish to mammals have been identified in the spacer domain by sequence alignment. Previous studies have shown that exosite 3 in the spacer domain plays a critical role for substrate recognition (Blood 115:2300–10, 2010), and modification of this exosite generates ADAMTS13 variants with improved specific activity but reduced autoantibody binding (Blood 119:3836–43, 2012). In the present study, using a site-directed mutagenesis approach, we identified a novel exosite near exosite 3 in the spacer domain, termed exosite 4, a region between residues Glu634 and Arg639. A partial (ΔEx4a:deletion of Leu632-Asp635 or ΔEx4b:deletion of Arg636-Arg639) or complete deletion of the exosite (ΔEx4) significantly impaired proteolytic activity towards peptidyl VWF73 and multimeric VWF. Moreover, substitution of all surface exposed residues in Ex4A (LTED/AAAA) or Ex4b (RLPR/AAAA) with alanine had a similarly detrimental effect on proteolytic activity. Further studies demonstrated that the residues Asp635 and Arg636 in exosite 4 play a critical role for substrate recognition. We conclude that the region between residues Glu634 and Arg639 is a novel exosite necessary for recognition and cleavage of VWF. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A site-directed mutagenesis study of human placental aromatase.

1992 ◽  
Vol 267 (2) ◽  
pp. 762-768
Author(s):  
D J Zhou ◽  
K R Korzekwa ◽  
T Poulos ◽  
S A Chen
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

scholarly journals A Tocqueville For the North? André Siegfried and Canada

2005 ◽  
Vol 14 (1) ◽  
pp. 117-136 ◽  
Author(s):  
Sean Kennedy
Keyword(s):  
Critical Role ◽  
Second World War ◽  
Traditional Values ◽  
French Canada ◽  
The North ◽  
Vichy Regime ◽  
The Face ◽  
A Site ◽  
French National Identity ◽  
Second World

Abstract This paper argues that André Siegfried’s writings on Canada played a critical role in shaping his vision of French national identity. Siegfried’s studies of Canada have long been praised for their insight, but recent scholarship has emphasized his role in promoting both anti-Americanism and an exclusionary vision of what it meant to be French during the first half of the twentieth century. For Siegfried, Canada represented a site of managed contestation between British and French culture but also an early example of the deleterious effects of Americanization. His problematic view of French Canada as essentially conservative and unchanging in the face of such challenges reinforced his conviction that France itself should remain true to “traditional” values. The exclusionary implications of his ideas were most evident when Siegfried appeared to accommodate himself to the Vichy regime, but they also persisted after the Second World War.

scholarly journals Variation of Two S3b Residues in KV4.1–4.3 Channels Underlies Their Different Modulations by Spider Toxin κ-LhTx-1

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhen Xiao ◽  
Piao Zhao ◽  
Xiangyue Wu ◽  
Xiangjin Kong ◽  
Ruiwen Wang ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Voltage Dependence ◽  
Underlying Mechanism ◽  
Voltage Sensor ◽  
Action Mode ◽  
Gating Modifier ◽  
Peptide Toxin ◽  
Peptide Toxins ◽  
Key Residues ◽  
Animal Venoms

The naturally occurred peptide toxins from animal venoms are valuable pharmacological tools in exploring the structure-function relationships of ion channels. Herein we have identified the peptide toxin κ-LhTx-1 from the venom of spider Pandercetes sp (the Lichen huntsman spider) as a novel selective antagonist of the KV4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded KV4 channels’ voltage sensor activation, and mutation analysis has confirmed its binding site on channels’ S3b region. Interestingly, κ-LhTx-1 differently modulated the gating of KV4 channels, as revealed by toxin inhibiting KV4.2/4.3 with much more stronger voltage-dependence than that for KV4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of KV4.1 in a much more stable resting state than that for KV4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between KV4.1(280FVPK283) and KV4.3(275VMTN278) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in KV4.1, D281 in KV4.2 and N278 in KV4.3 being the key residues. Furthermore, the last two residues in this segment of each KV4 channel (P282/K283 in KV4.1, T280/D281 in KV4.2 and T277/N278 in KV4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in KV4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on KV4 channels’ structure features. Moreover, κ-LhTx-1 might be useful in developing anti-arrhythmic drugs given its high affinity, high selectivity and unique action mode in interacting with the KV4.2/4.3 channels.

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