Peptide toxins isolated from animal venom secretions have proven to be useful pharmacological tools for probing the structure and function of a number of molecular receptors. Their molecular structures are stabilized by posttranslational formation of multiple disulfide bonds formed between sidechain thiols of cysteine residues, resulting in high thermal and chemical stability. Many of these peptides have been found to be potent modulators of ion channels, making them particularly influential in this field. Recently, several peptide toxins have been described that have an unusual tandem repeat organization, while also eliciting a unique pharmacological response toward ion channels. Most of these are two-domain peptide toxins from spider venoms, such as the double-knot toxin (DkTx), isolated from the Earth Tiger tarantula (Haplopelma schmidti). The unusual pharmacology of DkTx is its high avidity for its receptor (TRPV1), a property that has been attributed to a bivalent mode-of-action. DkTx has subsequently proven a powerful tool for elucidating the structural basis for the function of the TRPV1 channel. Interestingly, all tandem repeat peptides functionally characterized to date share this high avidity to their respective binding targets, suggesting they comprise an unrecognized structural class of peptides with unique structural features that result in a characteristic set of pharmacological properties. In this article, we explore the prevalence of this emerging class of peptides, which we have named Secreted, Cysteine-rich REpeat Peptides, or “SCREPs.” To achieve this, we have employed data mining techniques to extract SCREP-like sequences from the UniProtKB database, yielding approximately sixty thousand candidates. These results indicate that SCREPs exist within a diverse range of species with greatly varying sizes and predicted fold types, and likely include peptides with novel structures and unique modes of action. We present our approach to mining this database for discovery of novel ion-channel modulators and discuss a number of “hits” as promising leads for further investigation. Our database of SCREPs thus constitutes a novel resource for biodiscovery and highlights the value of a data-driven approach to the identification of new bioactive pharmacological tools and therapeutic lead molecules.

It has been proposed that a number of chemical-induced cell injuries result from disruption of the ability of the cell to control calcium. Many of the techniques used to develop this theory have relied on indirect measurements of intracellular calcium. The advent of digital imaging fluorescence microscopy has allowed a more direct examination of the relationship between calcium and cell damage. Results indicate that cytosolic calcium does not play a central role in the initiation of oxidative injury in a number of cell types. Changes in calcium homeostasis occur well after the appearance of other indications of cell injury. However, recent studies indicate that a mitochondrial lesion occurs relatively early in the time course of oxidative cell injury. Calcium may play a role in the development of this lesion.

The psychosocial impact of bariatric surgery has not been studied as diligently as the physical impact, particularly within the first 6 months following surgery. The aim of the present study was to explore psychosocial adjustment in UK bariatric candidates within this time-scale. Six female participants were purposively recruited to complete a semi-structured interview, and Interpretative Phenomenological Analysis was used to analyse their experiences. Four super-ordinate themes emerged from the interview data which were: (1) "It was me but it wasn’t me": pre-surgery identity, (2) "I don’t see myself as this fat blob of a person anymore": transforming identity, (3) "No easy road to weight loss": the challenges of living with stomach restriction, (4) "I’m letting people in more now": re-engaging with others and the world. Participant accounts highlighted a largely positive psychosocial experience following surgery. Results are discussed in support of previous literature and suggest (1) the exploration of identity more thoroughly, and (2) the importance of routine pre- and post-surgery psychosocial support to be incorporated as part of Tier 3 and 4 bariatric services.

1 The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image¯uorescence microscopy. 2 Con¯uent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ET A : 70% ET B ), with a density of approximately 3400+280 ET A and 8000+610 ET B receptors/cell (n=3 experiments). The density of ET B , but not ET A receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ET B receptor density such that the ET receptor subtype proportions were approximately equal (55% ET A ; 45% ET B ) and similar to those previously observed in intact rat tracheal smooth muscle. ] i increase was due primarily to the mobilization of IP 3 -sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ET B receptor activation was exclusively coupled to extracellular calcium in¯ux. 4 Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ET A receptors at a density of 6100+800 receptors cell 71 (n=3 experiments). Serum deprivation was without eect on either ET receptor subtype proportion or ET A receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentrationdependent increase in [Ca 2+ ] i (EC 50 : 15 nM), with a peak [Ca 2+ ] i increase to greater than 700 nM. Furthermore, the ET A -mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5 In summary, rat cultured tracheal airway smooth muscle cells contained both ET A and ET B receptors. ET A receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca 2+ from intracellular stores and a strong rise in [Ca 2+ ] i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ET B receptors increased signi®cantly during cell growth, an eect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ET B receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca 2+ ] i , which appeared to be attributable to the in¯ux of extracellular Ca 2+ . In contrast, the populations of ET receptors and their linkage to [Ca 2+ ] i were markedly dierent in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.

The possibility that significant changes in endothelin (ET)A- and ETB-receptor density and function occur in airway smooth muscle cells (ASMCs) during cell growth and extended cell culture was investigated in sheep tracheal ASMCs. As in intact tracheal smooth muscle tissue from this species, early-passage sheep ASMCs contained a homogeneous population of ETA receptors. However, growth of ASMCs from seeding to postconfluence and repeated passage of ASMCs (6th to 14th passages) was associated with a substantial increase in ETB-receptor density, with no change in ETA-receptor density. ET-1-induced stimulation of ETBreceptors increased the intracellular Ca2+ concentration in single ASMCs. Interestingly, a 2-day period of serum deprivation completely eliminated the increase in ETB-receptor density and the ETB receptor-mediated change in intracellular Ca2+ concentration. In summary, growth and repeated passage of sheep ASMCs were associated with a profound and selective increase in the density and function of the ETB receptor, a receptor subtype not present in early-passage ASMCs and not detected in intact sheep tracheal airway smooth muscle.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

No abstract

Receptor avidity through multivalency is a highly sought‐after property of ligands. While readily available in nature in the form of bivalent antibodies, this property remains challenging to engineer in synthetic molecules. The discovery of several bivalent venom peptides containing two homologous and independently folded domains (in a tandem repeat arrangement) has provided a unique opportunity to better understand the underpinning design of multivalency in multimeric biomolecules, as well as how naturally occurring multivalent ligands can be identified. In previous work, we classified these molecules as a larger class termed secreted cysteine‐rich repeat‐proteins (SCREPs). Here, we present an online resource; ScrepYard, designed to assist researchers in identification of SCREP sequences of interest and to aid in characterizing this emerging class of biomolecules. Analysis of sequences within the ScrepYard reveals that two‐domain tandem repeats constitute the most abundant SCREP domain architecture, while the interdomain “linker” regions connecting the functional domains are found to be abundant in amino acids with short or polar sidechains and contain an unusually high abundance of proline residues. Finally, we demonstrate the utility of ScrepYard as a virtual screening tool for discovery of putatively multivalent peptides, by using it as a resource to identify a previously uncharacterized serine protease inhibitor and confirm its predicted activity using an enzyme assay.