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The properties of specific binding site of 125I-radioiodinated myotoxin a, a novel Ca++ releasing agent, in skeletal muscle sarcoplasmic reticulum
Abstract
It was found that myotoxin a (MYTX), which is isolated from prairie rattlesnakes (Crotalus viridis viridis), is a powerful Ca++ releaser in the heavy fraction of sarcoplasmic reticulum (HSR). 125I-labeled MYTX (125I-MYTX), which has high Ca(++)-releasing ability, was successfully prepared. It specifically bound to a single class of binding sites in HSR with a KD of 0.4 microM and Bmax of 6 nmol/mg of protein. 125I-MYTX binding was markedly inhibited by Na+ and K+, whereas it was little affected by Ca++ and Mg++. The binding activity was markedly decreased by spermine, a blocker of Ca++ releasing channels, and was not affected by the other modulators of Ca++ release such as caffeine, procaine or ruthenium red. Spermine decreased the binding in a concentration-dependent manner with the IC50 value of 20 microM. Scatchard analysis of 125I-MYTX binding indicated that the Bmax value was decreased by spermine, although the KD value was not changed, which indicates a noncompetitive mode of inhibition. 125I-MYTX did not bind to the purified ryanodine receptor. Ca++ electrode experiments indicated that MYTX induced Ca++ release from HSR at doses of 0.1 microM or more; this was abolished by spermine. The maximal response to MYTX (10 microM) was further increased by caffeine (10 mM) in 45Ca++ release, which probably indicates that the effects of MYTX and caffeine are synergistic in Ca(++)-releasing action. These results suggest that MYTX binds to an important regulatory protein of Ca++ release, which is not the ryanodine receptor.
... AVPIAQK is derived from the N-terminus of the second mitochondria-derived activator of caspase (Smac) protein. Induction of caspase-3 activity was observed after the treatment of HeLa cells with SmacN7-K (FITC)-CyLoP-1 in comparison with K(FITC)-CyLoP-1 or even with SmacN7-K(FITC)-Tat (49)(50)(51)(52)(53)(54)(55)(56)(57), which is derived from Tat, one of the best characterized and explored CPPs. CyLoP-1 or Smac-N7, as well as the Tat derivative peptide, failed to increase the caspase-3 activity alone. ...
... MYTX is closely related to crotamine and also demonstrates CPP activity [50]. Several studies carried out in the 1980s and 1990s using radiolabeled MYTX showed that it induces Ca 2? release from the sarcoplasmic reticulum (SR) of skeletal muscle cells [51][52][53], probably by binding to calsequestrin [54], ATP/ ADP translocase [55] and inhibiting Ca 2? -ATPase [56]. The attachment of MYTX to Ca 2? -ATPase is believed to cause uncoupling of the calcium pump [56]. ...
Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides—a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.
... These assays showed that these toxins promote calcium release from the sarcoplasmic reticulum via the opening of the ryanodine receptor (RyR), as assessed using several pharmacological agents [39][40][41]. It has been speculated that crotamine binds to proteins associated with the RyR, such as calsequestrin [42], because no direct binding to the RyR was observed [43]. Our results ...
Crotamine is a rattlesnake-derived toxin that causes fast-twitch muscle paralysis. As a cell-penetrating polypeptide, crotamine has been investigated as an experimental anti-cancer and immunotherapeutic agent. We hypothesized that molecules targeting crotamine could be designed to study its function and intervene in its adverse activities. Here, we characterize synthetic crotamine and show that, like the venom-purified toxin, it induces hindlimb muscle paralysis by affecting muscle contraction and inhibits KCNA3 (Kv1.3) channels. Synthetic crotamine, labeled with a fluorophore, displayed cell penetration, subcellular myofiber distribution, ability to induce myonecrosis, and bind to DNA and heparin. Here, we used this functionally validated synthetic polypeptide to screen a combinatorial phage display library for crotamine-binding cyclic peptides. Selection for tryptophan-rich peptides was observed, binding of which to crotamine was confirmed by ELISA and gel shift assays. One of the peptides (CVWSFWGMYC), synthesized chemically, was shown to bind both synthetic and natural crotamine and to block crotamine-DNA binding. In summary, our study establishes a functional synthetic substitute to the venom-derived toxin and identifies peptides that could further be developed as probes to target crotamine.
Key messages
Synthetic crotamine was characterized as a functional substitute for venom-derived crotamine based on myotoxic effects.
A combinatorial peptide library was screened for crotamine-binding peptides.
Tryptophan-rich peptides were shown to bind to crotamine and interfere with its DNA binding.
Crotamine myofiber distribution and affinity for tryptophan-rich peptides provide insights on its mechanism of action.
... Da es sich hierbei um einen TTX-sensitiven Effekt zu handeln scheint, wird vermutet, dass diese Wirkungsweise von Crotamin über entsprechende Natriumkanäle der Plasmamembran vermittelt wird (Filho et al. 1978;Tsai et al. 1981;Hong et al. 1985;Brazil et al. 1993). (Furukawa et al. 1994;Ohkura et al. 1994;Ohkura et al. 1995). ...
Für das Verständnis der pharmakologischen Aktivität von Proteinen und Peptiden ist es wichtig, den Zusammenhang zwischen Molekülstruktur und Wirkungsweise zu untersuchen. Schlangengifte setzen sich aus einer Vielzahl von Komponenten zusammen, die in unterschiedlichster Weise den Organismus der Beutetiere beeinflussen und schädigen. Sie stellen daher eine reiche Quelle für Substanzen mit biologischer Wirksamkeit dar. Die Isolierung der einzelnen Komponenten bietet somit eine effektive Möglichkeit, pharmakologisch aktive Proteine und Peptide zu gewinnen. Der Fokus der vorliegenden Arbeit lag auf der Isolierung und Charakterisierung von Einzelkomponenten aus dem Gift der südamerikanischen Klapperschlange Crotalus durissus terrificus. Es konnten zwei der Hauptbestandteile, Crotamin, sowie eine Mischung an Crotoxin B Isoformen, mittels RP-HPLC isoliert und durch massenspektrometrische Untersuchungen identifiziert werden. Im Weiteren lag der Schwerpunkt auf der Charakterisierung der Interaktion von Crotamin mit künstlichen Membranen. Crotamin ist ein amphiphatisches, hoch basisches Polypeptid mit einer Größe von 4,9 kDa und stark positiver Oberflächenladung. Zunächst als Neurotoxin beschrieben, sind für Crotamin mittlerweile eine Vielzahl weiterer Wirkungen berichtet worden. Zu diesen gehören myotoxische, analgetische, antimikrobielle und antitumorale Effekte. Die Wirkungsweise beruht teilweise auf einer direkten Interaktion mit den Lipiden der Membran. Crotamin werden dabei sowohl membranbeeinflussende Eigenschaften, als auch die Fähigkeit zur Translokation in die Zelle zugeschrieben. Die vorliegende Arbeit zeigt deutlich den Einfluss der Lipidkomposition von Membranen auf die membranbeeinflussenden Eigenschaften von Crotamin. In Monolayern aus Asolektin führte die Anwesenheit von Cholesterin zu einem langsameren Einbau von Crotamin. Zudem verursachte sowohl die Anwesenheit bivalenter Kationen, als auch die Erhöhung des lateralen Drucks im Monolayer, einen beschleunigten Einbau von Crotamin. Des Weiteren konnte gezeigt werden, dass Crotamin die Fluidität von Vesikelmembranen verändert. Bei reinen Asolektinvesikeln führte Crotamin zu festeren, bei DOPC-Vesikeln zu fluideren Membranen. Bei Vesikeln, deren Membran zusätzlich Cholesterin enthielt, führte Crotamin unabhängig von der gewählten Grundsubstanz (Asolektin oder DOPC) zu fluideren Membranen. Das Ausmaß der Fluidisierung war jedoch abhängig von der jeweiligen Cholesterinkonzentration. Je mehr Cholesterin die Vesikelmembranen enthielten, desto geringer die Wirkung von Crotamin. Generell war hierbei der Einfluss der Cholesterinkonzentration bei asolektinhaltigen Vesikeln größer als bei DOPC-haltigen. Demnach wirkte Cholesterin den membranbeeinflussenden Eigenschaften von Crotamin entgegen. Vermutlich beruhte dies unter anderem auf der geringeren Fluidität von cholesterinhaltigen Membranen. Zudem könnte für die polaren Kopfgruppen der Lipide die Wahrscheinlichkeit zur Interaktion mit kationischen Molekülen wie Crotamin durch die Anwesenheit von Cholesterin verringert sein. Der stärkere Einfluss von Crotamin auf die Fluidität von asolektinhaltigen Membranen könnte auf den, in Asolektin enthaltenen, Lipiden mit negativer Ladung beruhen. Diese könnten, auf Grund einer negativeren Oberflächenladung der Membran, zu einer stärkeren Anziehung von Crotamin führen. Außerdem könnte Crotamin, durch die Interaktion mit negativ geladenen Lipiden, die Bildung von Domänen verursachen. Die Untersuchungen dieser Arbeit lassen somit den Schluss zu, dass sowohl Cholesterin als auch Lipide mit negativer Ladung die Wirkungsweise von Crotamin auf Membranen verändern. Zudem wird sie durch die Anwesenheit bivalenter Kationen in der umgebenden Lösung und dem lateralen Membrandruck beeinflusst. Darüber hinaus konnte gezeigt werden, dass Crotamin die Kalziumhomöostase von neuronalen Zellen beeinflusst. Hierbei verursachte Crotamin einen konzentrationsabhängigen Anstieg der intrazellulären freien Kalziumkonzentration.
Numerous neurotoxins that alter Na⁺-channel function have been shown to be useful tools for characterizing Na⁺ channels. Polypeptide blockers of voltage-dependent K⁺ channels (dendrotoxins, etc.) and Ca²⁺-activated K⁺ channels (apamine, etc.) have been studied extensively by numerous investigators. Peptide toxins, calciseptine and ω-conotoxins have been attracting much attention as inhibitors of L-type and N-type Ca²⁺ channels, respectively, while ω-conotoxins-MVIIC and ω-agatoxin IVA have been used as new types of Ca²⁺-channel blockers. Ryanodine and bromoeudistomin D analogues have been extensively used to elucidate Ca²⁺-release-channel functions and to purify its target protein. Polypeptide toxins (myotoxin a, etc.) and macrolides (FK 506, etc.) are useful Ca²⁺ releasers with a novel mechanism, while natural products such as thapsigargin and gingerol have been used as modulators of Ca²⁺-pumping ATPase. Some modulators of the function of myosin (purealin, etc.) and actin (goniodomin A, etc.) have been demonstrated to be important chemical probes for understanding the physiological roles of the contractile proteins in structural changes and their interaction in muscle contraction. A large number of protein kinase inhibitors (staurosporine, etc.) and phosphatase inhibitors (okadaic acid, etc.) are widely used as first-choice reagents for studying protein phosphorylation. These natural products have become essential tools for studying the regulatory mechanism of cellular ion movements, muscle contraction and protein phosphorylation.
In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the rime to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 mu M PIP2 were not modified by ruthenium red. PIP2 (>0.1 mu M) markedly accelerated the rate of Ca-45(2+) efflux from SR vesicles in a concentration-dependent manner. The PIP2-induced Ca-45(2+) efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced Ca-45(2+) release was clearly different from that in the Ca2+-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [H-3]ryanodine or [H-3]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca2+-induced Ca2+ release channels (the ryanodine receptor 1).
The ryanodine receptor (RyR) is an essential element in excitation–contraction coupling. Ca2+ release from the sarcoplasmic reticulum (SR) is required for skeletal and heart muscle contraction. The inositol-1,4,5-triphosphate-receptor (IP3R) plays an important function in the signal transduction of many hormones, cytokines, growth factors and antigens, and in fertilisation. Modulators of intracellular calcium channels are used for the treatment of malignant hyperthermia associated with abnormal Ca2+ transport and may be applied in the treatment of cardiovascular and neurodegenerative disorders.
Abstract Myotoxin a and a group of closely related, small, basic toxins cause myonecrotic destruction of muscle tissue upon envenomation. The sarcoplasmic reticulum swells and eventually breaks down to small vesicles. Degeneration of myofibrils and myofilaments ensues and loss of the classic striation pattern is apparent. These toxins exhibit high sequence similarity as well as sequence microheterogeneity. A conformational heterogeneity was recently discovered in myotoxin a. The existence of myotoxin a in two forms in equilibrium in solution hinders the generation of a well defined three-dimensional structure. The difference, if any, in the biological activity of the two forms has not been established yet. Recent biochemical studies indicate that myotoxin a is a potent Ca2+ releasing agent that binds to calsequestrin in the lumen of the sarcoplasmic reticulum.
Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120−1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181−1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.
Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 degrees C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 degrees C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 degrees C.
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