Isolation and structural identification of a potassium ion channel Kv4.1 inhibitor SsTx-P2 from centipede venom. / 蜈蚣毒液中钾离子通道Kv4.1抑制剂SsTx-P2的分离和结构鉴定.

OBJECTIVES: To isolate a potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its sequence and structure. METHODS: Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with matrix assisted laser desorption ionization-time-of-flight mass spectrometry; its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry; its structure was established based on iterative thread assembly refinement online analysis. RESULTS: A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues NH2-ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 µmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its structure showed that SsTx-P2 shared a conserved helical structure. CONCLUSIONS: The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1 and displays structural conservation.

Molecular mechanism by which spider-driving peptide potentiates coagulation factors.

Hemostasis is a crucial process that quickly forms clots at injury sites to prevent bleeding and infections. Dysfunctions in this process can lead to hemorrhagic disorders, such as hemophilia and thrombocytopenia purpura. While hemostatic agents are used in clinical treatments, there is still limited knowledge about potentiators targeting coagulation factors. Recently, LCTx-F2, a procoagulant spider-derived peptide, was discovered. This study employed various methods, including chromogenic substrate analysis and dynamic simulation, to investigate how LCTx-F2 enhances the activity of thrombin and FXIIa. Our findings revealed that LCTx-F2 binds to thrombin and FXIIa in a similar manner, with the N-terminal penetrating the active-site cleft of the enzymes and the intermediate section reinforcing the peptide-enzyme connection. Interestingly, the C-terminal remained at a considerable distance from the enzymes, as evidenced by the retention of affinity for both enzymes using truncated peptide T-F2. Furthermore, results indicated differences in the bonding relationship of critical residues between thrombin and FXIIa, with His13 facilitating binding to thrombin and Arg7 being required for binding to FXIIa. Overall, our study sheds light on the molecular mechanism by which LCTx-F2 potentiates coagulation factors, providing valuable insights that may assist in designing drugs targeting procoagulation factors.

Chronic cough relief by allosteric modulation of P2X3 without taste disturbance.

P2X receptors are cation channels that sense extracellular ATP. Many therapeutic candidates targeting P2X receptors have begun clinical trials or acquired approval for the treatment of refractory chronic cough (RCC) and other disorders. However, the present negative allosteric modulation of P2X receptors is primarily limited to the central pocket or the site below the left flipper domain. Here, we uncover a mechanism of allosteric regulation of P2X3 in the inner pocket of the head domain (IP-HD), and show that the antitussive effects of quercetin and PSFL2915 (our nM-affinity P2X3 inhibitor optimized based on quercetin) on male mice and guinea pigs were achieved by preventing allosteric changes of IP-HD in P2X3. While being therapeutically comparable to the newly licensed P2X3 RCC drug gefapixant, quercetin and PSFL2915 do not have an adverse effect on taste as gefapixant does. Thus, allosteric modulation of P2X3 via IP-HD may be a druggable strategy to alleviate RCC.

Molecular mechanism of HNTX-I activates the intermediate-conductance Ca2+-activated K+ (IK) channels.

The IK channel, a potassium ion channel regulated by calcium ions and voltages in a bidirectional manner, has been implicated in a range of diseases. However, there are currently few compounds available that can target the IK channel with high potency and specificity. Hainantoxin-I (HNTX-I) is the first peptide activator of IK channel discovered so far, but its activity is not ideal, and the underlying mechanism interaction between HNTX-I toxin and IK channel remains unclear. Thus, our study aimed to enhance the potency of IK channel activating peptides derived from HNTX-I and elucidate the molecular mechanism underlying the interaction between HNTX-I and the IK channel. By employing virtual alanine scanning mutagenesis, we generated 11 HNTX-I mutants using site-directed mutagenesis to pinpoint specific residues crucial for the HNTX-I and IK channel interaction. Subsequently, we identified key residues on the IK channel that are involved in the interaction with HNTX-I. Additionally, molecular docking was employed to guide the molecular engineering process and clarify the binding interface between HNTX-I and the IK channel. Our results demonstrate that HNTX-I primarily acts on the IK channel via the N-terminal amino acid, and its interaction with the IK channel is mediated by electrostatic and hydrophobic interactions, specifically the amino acid residues at positions 1, 3, 5, and 7 on HNTX-I. This study provides valuable insights into the peptide toxins that may serve as potential templates for the development of activators with enhanced potency and selectivity for the IK channel.

General mechanism of spider toxin family I acting on sodium channel Nav1.7.

Various peptide toxins in animal venom inhibit voltage-gated sodium ion channel Nav1.7, including Nav-targeting spider toxin (NaSpTx) Family I. Toxins in NaSpTx Family I share a similar structure, i.e., N-terminal, loops 1-4, and C-terminal. Here, we used Mu-theraphotoxin-Ca2a (Ca2a), a peptide isolated from Cyriopagopus albostriatus, as a template to investigate the general properties of toxins in NaSpTx Family I. The toxins interacted with the cell membrane prior to binding to Nav1.7 via similar hydrophobic residues. Residues in loop 1, loop 4, and the C-terminal primarily interacted with the S3-S4 linker of domain II, especially basic amino acids binding to E818. We also identified the critical role of loop 2 in Ca2a regarding its affinity to Nav1.7. Our results provide further evidence that NaSpTx Family I toxins share similar structures and mechanisms of binding to Nav1.7.

Anticarin-ß shows a promising anti-osteosarcoma effect by specifically inhibiting CCT4 to impair proteostasis.

Unlike healthy, non-transformed cells, the proteostasis network of cancer cells is taxed to produce proteins involved in tumor development. Cancer cells have a higher dependency on molecular chaperones to maintain proteostasis. The chaperonin T-complex protein ring complex (TRiC) contains eight paralogous subunits (CCT1-8), and assists the folding of as many as 10% of cytosolic proteome. TRiC is essential for the progression of some cancers, but the roles of TRiC subunits in osteosarcoma remain to be explored. Here, we show that CCT4/TRiC is significantly correlated in human osteosarcoma, and plays a critical role in osteosarcoma cell survival. We identify a compound anticarin-ß that can specifically bind to and inhibit CCT4. Anticarin-ß shows higher selectivity in cancer cells than in normal cells. Mechanistically, anticarin-ß potently impedes CCT4-mediated STAT3 maturation. Anticarin-ß displays remarkable antitumor efficacy in orthotopic and patient-derived xenograft models of osteosarcoma. Collectively, our data uncover a key role of CCT4 in osteosarcoma, and propose a promising treatment strategy for osteosarcoma by disrupting CCT4 and proteostasis.

Extracellular domain of PepT1 interacts with TM1 to facilitate substrate transport.

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.

Shrew's venom quickly causes circulation disorder, analgesia and hypokinesia.

Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit.

A Buthus martensii Karsch scorpion sting targets Nav1.7 in mice and mimics a phenotype of human chronic pain.

ABSTRACT: Gain-of-function and loss-of-function mutations in Nav1.7 cause chronic pain and pain insensitivity, respectively. The preferential expression of Nav1.7 in the peripheral nervous system and its role in human pain signaling make Nav1.7 a promising target for next-generation pain therapeutics. However, pharmacological agents have not fully recapitulated these pain phenotypes, and because of the lack of subtype-selective molecular modulators, the role of Nav1.7 in the perception of pain remains poorly understood. Scorpion venom is an excellent source of bioactive peptides that modulate various ion channels, including voltage-gated sodium (Nav) channels. Here, we demonstrate that Buthus martensii Karsch scorpion venom (BV) elicits pain responses in mice through direct enhancement of Nav1.7 activity and have identified Makatoxin-3, an α-like toxin, as a critical component for BV-mediated effects on Nav1.7. Blocking other Nav subtypes did not eliminate BV-evoked pain responses, supporting the pivotal role of Nav1.7 in BV-induced pain. Makatoxin-3 acts on the S3-S4 loop of voltage sensor domain IV (VSD4) of Nav1.7, which causes a hyperpolarizing shift in the steady-state fast inactivation and impairs inactivation kinetics. We also determined the key residues and structure-function relationships for the toxin-channel interactions, which are distinct from those of other well-studied α toxins. This study not only reveals a new mechanism underlying BV-evoked pain but also enriches our knowledge of key structural elements of scorpion toxins that are pivotal for toxin-Nav1.7 interactions, which facilitates the design of novel Nav1.7 selective modulators.

A designed antifungal peptide with therapeutic potential for clinical drug-resistant Candida albicans.

Due to the increasing drug-resistant of Candida albicans (C. albicans), there is an urgent need to develop a novel therapeutic agent for C. albicans induced inflammatory disease treatment. Antimicrobial peptides (AMPs) are regarded as one of the most promising antifungal drugs. However, most of the designed AMPs showed side-effects. In the present study, 10 novel peptides were designed based on the sequence of frog skin secretions peptide (Ranacyclin AJ). Among them, AKK8 (RWRFKWWKK) exhibited the strongest antifungal effect against both standard and clinically isolated drug-resistant C. albicans. AKK8 killed C. albicans (within 30 min), and the antifungal effect lasted for 24 h, showed an efficient and long lasted antifungal effect against C. albicans. Notably, AKK8 showed low toxicity to human red blood cells and high stability in human serum. Moreover, AKK8 administration showed therapeutic effects on systemic infections mice induced by the clinical drug-resistant C. albicans, in a dose-depended manner. These findings suggested that AKK8 may be a potential candidate for the anti-inflammation treatments for diseases caused by clinical drug-resistant C. albicans.

The Functional Role of Voltage-Gated Sodium Channel Nav1.5 in Metastatic Breast Cancer.

Voltage-gated sodium channels (VGSCs), which are abnormally expressed in various types of cancers such as breast cancer, prostate cancer, lung cancer, and cervical cancer, are involved in the metastatic process of invasion and migration. Nav1.5 is a pore-forming α subunit of VGSC encoded by SCN5A. Various studies have demonstrated that Nav1.5, often as its neonatal splice form, is highly expressed in metastatic breast cancer cells. Abnormal activation and expression of Nav1.5 trigger a variety of cellular mechanisms, including changing H+ efflux, promoting epithelial-to-mesenchymal transition (EMT) and the expression of cysteine cathepsin, to potentiate the metastasis and invasiveness of breast cancer cells in vitro and in vivo. Here, we systematically review the latest available data on the pro-metastatic effect of Nav1.5 and its underlying mechanisms in breast cancer. We summarize the factors affecting Nav1.5 expression in breast cancer cells, and discuss the potential of Nav1.5 blockers serving as candidates for breast cancer treatment.

LCTX-F2, a Novel Potentiator of Coagulation Factors From the Spider Venom of Lycosa singoriensis.

Spider venoms contain many functional proteins/peptides such as proteinases, serine/cysteine proteinase inhibitors, insecticidal toxins, and ion channel toxins. However, to date, no peptide toxin with procoagulant activities has been identified from spider venom. In this study, a novel toxin LCTX-F2 with coagulation-promoting activity was identified and characterized in the venom of the spider Lycosa singoriensis (L. singoriensis). LCTX-F2 significantly shortened activated partial thromboplastin time (APTT), clotting time, and plasma recalcification time. This toxin directly interacted with several coagulation factors such as FXIIa, kallikrein, thrombin, and FXa and increased their protease activities. In liver bleeding and tail bleeding mouse models, LCTX-F2 significantly decreased the number of blood cells and bleeding time in a dose-dependent manner. At the same dosage, LCTX-F2 exhibited a more significant procoagulant effect than epsilon aminocaproic acid (EACA). Moreover, LCTX-F2 showed no cytotoxic or hemolytic activity against either normal cells or red blood cells. Our results suggested that LCTX-F2 is a potentiator of coagulation factors with the potential for use in the development of procoagulant drugs.

TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in membrane potential.

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

Identification and Characterization of ShSPI, a Kazal-Type Elastase Inhibitor from the Venom of Scolopendra Hainanum.

Elastase is a globular glycoprotein and belongs to the chymotrypsin family. It is involved in several inflammatory cascades on the basis of cleaving the important connective tissue protein elastin, and is strictly regulated to a balance by several endogenous inhibitors. When elastase and its inhibitors are out of balance, severe diseases will develop, especially those involved in the cardiopulmonary system. Much attention has been attracted in seeking innovative elastase inhibitors and various advancements have been taken on clinical trials of these inhibitors. Natural functional peptides from venomous animals have been shown to have anti-protease properties. Here, we identified a kazal-type serine protease inhibitor named ShSPI from the cDNA library of the venom glands of Scolopendra hainanum. ShSPI showed significant inhibitory effects on porcine pancreatic elastase and human neutrophils elastase with Ki values of 225.83 ± 20 nM and 12.61 ± 2 nM, respectively. Together, our results suggest that ShSPI may be an excellent candidate to develop a drug for cardiopulmonary diseases.

An Antiviral Peptide from Alopecosa nagpag Spider Targets NS2B-NS3 Protease of Flaviviruses.

Flaviviruses are single-stranded RNA viruses predominantly transmitted by the widely distributed Aedes mosquitoes in nature. As important human pathogens, the geographic reach of Flaviviruses and their threats to public health are increasing, but there is currently no approved specific drug for treatment. In recent years, the development of peptide antivirals has gained much attention. Natural host defense peptides which uniquely evolved to protect the hosts have been shown to have antiviral properties. In this study, we firstly collected the venom of the Alopecosa nagpag spider from Shangri-La County, Yunnan Province. A defense peptide named Av-LCTX-An1a (Antiviral-Lycotoxin-An1a) was identified from the spider venom, and its anti-dengue serotype-2 virus (DENV2) activity was verified in vitro. Moreover, a real-time fluorescence-based protease inhibition assay showed that An1a functions as a DENV2 NS2B-NS3 protease inhibitor. Furthermore, we also found that An1a restricts zika virus (ZIKV) infection by inhibiting the ZIKV NS2B-NS3 protease. Together, our findings not only demonstrate that An1a might be a candidate for anti-flavivirus drug but also indicate that spider venom is a potential resource library rich in antiviral precursor molecules.

JZTX-V Targets the Voltage Sensor in Kv4.2 to Inhibit Ito Potassium Channels in Cardiomyocytes.

Kv4 potassium channels are responsible for transient outward K+ currents in the cardiac action potential (AP). Previous experiments by our group demonstrated that Jingzhaotoxin-V (JZTX-V) selectively inhibits A-type potassium channels. However, the specific effects of JZTX-V on the transient outward (Ito) current of cardiomyocytes and underlying mechanism of action remain unclear. In the current study, 100 nM JZTX-V effectively inhibited the Ito current and extended the action potential duration (APD) of neonatal rat ventricular myocytes (NRVM). We further analyzed the effects of JZTX-V on Kv4.2, a cloned channel believed to underlie the Ito current in rat cardiomyocytes. JZTX-V inhibited the Kv4.2 current with a half-maximal inhibitory concentration (IC50) of 13 ± 1.7 nM. To establish the molecular mechanism underlying the inhibitory action of JZTX-V on Kv4.2, we performed alanine scanning mutagenesis of Kv4.2 and JZTX-V and assessed the effects of the mutations on binding activities of the proteins. Interestingly, the Kv4.2 mutations V285A, F289A, and V290A reduced the affinity for JZTX-V while I275A and L277A increased the affinity for JZTX-V. Moreover, mutation of positively charged residues (R20 and K22) of JZTX-V and the hydrophobic patch (formed by W5, M6, and W7) led to a significant reduction in toxin sensitivity, indicating that the hydrophobic patch and electrostatic interactions played key roles in the binding of JZTX-V with Kv4.2. Data from our study have shed light on the specific roles and molecular mechanisms of JZTX-V in the regulation of Ito potassium channels and supported its utility as a potential novel antiarrhythmic drug.

Region-resolved proteomics profiling of monkey heart.

Nonhuman primates (NHPs) play an indispensable role in biomedical research because of their similarities in genetics, physiological, and neurological function to humans. Proteomics profiling of monkey heart could reveal significant cardiac biomarkers and help us to gain a better understanding of the pathogenesis of heart disease. However, the proteomic study of monkey heart is relatively lacking. Here, we performed the proteomics profiling of the normal monkey heart by measuring three major anatomical regions (vessels, valves, and chambers) based on iTRAQ-coupled LC-MS/MS analysis. Over 3,200 proteins were identified and quantified from three heart tissue samples. Furthermore, multiple bioinformatics analyses such as gene ontology analysis, protein-protein interaction analysis, and gene-diseases association were used to investigate biological network of those proteins from each area. More than 60 genes in three heart regions are implicated with heart diseases such as hypertrophic cardiomyopathy, heart failure, and myocardial infarction. These genes associated with heart disease are mainly enriched in citrate cycle, amino acid degradation, and glycolysis pathway. At the anatomical level, the revelation of molecular characteristics of the healthy monkey heart would be an important starting point to investigate heart disease. As a unique resource, this study can serve as a reference map for future in-depth research on cardiac disease-related NHP model and novel biomarkers of cardiac injury.

The defensive system of tree frog skin identified by peptidomics and RNA sequencing analysis.

The diversity of defensive peptides from skin of amphibians has been demonstrated. These peptides may have resulted from the diversity of microorganisms encountered by amphibians. In this study, peptidomics and RNA sequencing analyses were used to study deeply the defensive peptides of the skin secretions from Polypedates megacephalus. A total of 99 defensive peptides have been identified from the skin secretions. Among these peptides, 3 peptides were myotropical peptides and 34 peptides classified as protease inhibitor peptides. 5 lectins, 8 antimicrobial peptides, 26 immunomodulatory peptides, 10 wound-healing peptides and 13 other bioactive peptides were identified as belonging to the innate immune system. One antimicrobial peptide Pm-amp1 showed high similarity to antimicrobial peptide marcin-18. This peptide was successfully expressed and showed moderate activity against four tested strains. These identified peptides highlight the extensive diversity of defensive peptides and provide powerful tools to understand the defense weapon of frog.

PPIP: Automated Software for Identification of Bioactive Endogenous Peptides.

Endogenous peptides play an important role in multiple biological processes in many species. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is an important technique for detecting these peptides on a large scale. We present PPIP, which is a dedicated peptidogenomics software for identifying endogenous peptides based on peptidomics and RNA-Seq data. This software automates the de novo transcript assembly based on RNA-Seq data, construction of a protein reference database based on the de novo assembled transcripts, peptide identification, function analysis, and HTML-based report generation. Different function components are integrated using Docker technology. The Docker image of PPIP is available at https://hub.docker.com/r/shawndp/ppip , and the source code under GPL-3 license is available at https://github.com/Shawn-Xu/PPIP . A user manual of PPIP is available at https://shawn-xu.github.io/PPIP .