Novel Kunitz-like peptides discovered in zoanthid Palythoa caribaeorum through transcriptome sequencing

OBJECTIVE Palythoa caribaeorum(class Anthozoa) is a zoanthid that together jellyfishes,hydra,and sea anemones,which are venomous and predatory,belongs to the Phyllum Cnidaria.The distinguished feature in these marine animals is the cnidocytes in the body tissues,responsible for toxin production and injection that are used majorly for prey capture and defense.With exception for other anthozoans,the toxin cocktails of zoanthids have been scarcely studied and are poorly known.METHODS Based on the analysis of P.caribaeorum transcriptome,numerous predicted venom-featured polypeptides were identified,including allergens,neuro-toxins,membrane-active and Kunitz-like peptides(PcKuz).The three predicted PcKuz isotoxins(1 to 3) were selected for functional studies.Through computational processing comprising structural phylogenetic analysis,molecular docking,and dynamics simulation,PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor.RESULTS PcKuz3 fitted well as new functional Kunitz-type toxins with strong anti-locomotor activity as in vivo assessed in zebrafish larvae,with weak inhibitory effect toward proteases,as evaluated in vitro.Notably,PcKuz3 can suppress,at low concentration,the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish,which indicated PcKuz3 may have a neuroprotective effect.CONCLUSION Taken together,PcK uz3 figures as a novel neurotoxin structure which differs from known homologous peptides expressed in sea anemone.Moreover,the novel PcKuz3 provides an insightful hint for bio-drug development for prospective neurodegenerative disease treatment.

Structural and functional characterization of a novel ShK peptide and its analogue from zoanthids (corals)

OBJECTIVE To identify and characterize a novel neuroprotective ShK peptide and its analogue originated from coral P.caribaeoru.METHODS P.caribaeoru was collected and subjected to transcriptome sequencing at which further bioinformatics analysis had identified a unigene encoding a putative ShK protein candidate,named as PcShK1.PcShK1 and its rhodamine derivative(PcShK1-RhoB) were synthesized and tested for the neuroprotective effect in 6-OHDA induced Parkinson disease models in vitro and in vivo.Briefly,zebrafish larvae were co-exposed to 6-OHDA and various doses of the peptides;then,dopaminergic(DA) neurons immunoreactivity and locomotion behavior of the zebrafish larvae were examined.Similarly,PC12 cells were cultured with 6-OHDA in the absence or presence of different concentrations of peptides.Cell viability was determined by MTT assay while calcium flow in the PC12 cells was monitored by Fluo-4 fluorescent dye.RESULTS Compared with control group,6-OHDA treatment could lead to DA neurons loss and locomotion deficits in PD model of zebrafish larvae(P<0.01).Both PcS hK 1(2.5,5.0 and 7.5 μmol·L^(-1)) and PcS hK 1-RhoB(0.50 and 0.75 μmol·L^(-1)) were found to protect and restore dopaminergic neurons from6-OHDA mediated injury and locomotion deficiency in the PD zebrafish respectively(P<0.01).In addition,PcShK1(2.5 to 20.0 μmol·L^(-1)) and PcShK1-RhoB(0.6 to 2.5 μmol·L^(-1)) effectively prevented against 6-OHDA toxicity in PC12 cells(P<0.01).Further study revealed that they might exert their neuroprotective effects through regulating the calcium homeostasis.CONCLUSION PcS hK 1 and PcS hK 1-RhoB show neuroprotective effects on 6-OHDA induced PD models,and the underlying protective mechanisms of these peptides probably involve calcium homeostasis regulation.

Synergistic Effects of Chuanxiong-Chishao Herb-Pair on Promoting Angiogenesis at Network Pharmacological and Pharmacodynamic Levels

Objective： To investigate the synergistic effects of Chuanxiong-Chishao herb-pair （CCHP） on promoting angiogenesis in silico and in vivo. Methods： The mechanisms of action of an herb-pair, Chuanxiong- Chishao, were investigated using the network pharmacological and pharmacodynamic strategies involving computational drug target prediction and network analysis, and experimental validation. A set of network pharmacology methods were created to study the herbs in the context of targets and diseases networks, including prediction of target profiles and pharmacological actions of main active compounds in Chuanxiong and Chishao. Furthermore, the therapeutic effects and putative molecular mechanisms of Chuanxiong-Chishao actions were experimentally validated in a chemical-induced vascular insufficiency model of transgenic zebrafish in vivo. The mRNA expression of the predicted targets were further analyzed by real-time polymerase chain reaction （RT-PCR）. Results： The computational prediction results found that the compounds in Chuanxiong have antithrombotic, antihypertensive, antiarrhythmic, and antiatherosclerotic activities, which were closely related to protecting against hypoxic-ischemic encephalopathy, ischemic stroke, myocardial infarction and heart failure. In addition, compounds in Chishao were found to participate in anti-inflammatory effect and analgesics. Particularly, estrogen receptor α （ESR α） and hypoxia-inducible factor 1-α （HIF-1 α） were the most important potential protein targets in the predicted results. In vivo experimental validation showed that post-treatment of tetramethylpyrazine hydrochloride （TMpoHCI） and paeoniflorin （PF） promoted the regeneration of new blood vessels in zebrafish involving up-regulating ESR α mRNA expression. Co-treatment of TMP·HCI and PF could enhance the vessel sprouting in chemical-induced vascular insufficiency zebrafish at the optimal compatibility proportion of PF 10 μ mol/L with TMP·HCI 1 μ mol/L. Conclusions： The network pharmacological strategies combining drug target prediction and network analysis identified some putative targets of CCHP. Moreover, the transgenic zebrafish experiments demonstrated that the Chuanxiong-Chishao combination synergistically promoted angiogenic activity, probably involving ESR α signaling pathway.