RESUMEN
Sea anemones are venomous animals that rely on their venom for prey capture, defense against predators, and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analysis was used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electromechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species and little similarity with the secreted venom of closely related species. Overall, this demonstrates that regionalized and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.
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Proteoma , Anémonas de Mar , Animales , Anémonas de Mar/genética , Venenos de Cnidarios/genética , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Neurotoxinas/genética , Filogenia , ProteómicaRESUMEN
Although multiple approaches for characterizing protein-ligand interactions are available in target-based drug discovery, their throughput for determining selectivity is quite limited. Herein, we describe the application of native mass spectrometry for rapid, multiplexed screening of the selectivity of eight small-molecule ligands for five fatty acid-binding protein isoforms. Using high-resolution mass spectrometry, we were able to identify and quantify up to 20 different protein species in a single spectrum. We show that selectivity profiles generated by native mass spectrometry are in good agreement with those of traditional solution-phase techniques such as isothermal titration calorimetry and fluorescence polarization. Furthermore, we propose strategies for effective investigation of selectivity by native mass spectrometry, thus highlighting the potential of this technique to be used as an orthogonal method to traditional biophysical approaches for rapid, multiplexed screening of protein-ligand complexes.
RESUMEN
Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.
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Anémonas de Mar , Animales , Anémonas de Mar/química , Péptidos/farmacología , Péptidos/química , Canales de Potasio/metabolismo , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (NaV, TRPV1, KV and CaV). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly 13C,15N-labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch-clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (NaV) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (â¼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel.
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Anémonas de Mar , Animales , Humanos , Anémonas de Mar/química , Proteómica , Drosophila melanogaster/metabolismo , Australia , Péptidos/química , Disulfuros , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The voltage-gated potassium channel KV1.3 is an important therapeutic target for the treatment of autoimmune and neuroinflammatory diseases. The recent structures of KV1.3, Shaker-IR (wild-type and inactivating W434F mutant) and an inactivating mutant of rat KV1.2-KV2.1 paddle chimera (KVChim-W362F+S367T+V377T) reveal that the transition of voltage-gated potassium channels from the open-conducting conformation into the non-conducting inactivated conformation involves the rupture of a key intra-subunit hydrogen bond that tethers the selectivity filter to the pore helix. Breakage of this bond allows the side chains of residues at the external end of the selectivity filter (Tyr447 and Asp449 in KV1.3) to rotate outwards, dilating the outer pore and disrupting ion permeation. Binding of the peptide dalazatide (ShK-186) and an antibody-ShK fusion to the external vestibule of KV1.3 narrows and stabilizes the selectivity filter in the open-conducting conformation, although K+ efflux is blocked by the peptide occluding the pore through the interaction of ShK-Lys22 with the backbone carbonyl of KV1.3-Tyr447 in the selectivity filter. Electrophysiological studies on ShK and the closely-related peptide HmK show that ShK blocks KV1.3 with significantly higher potency, even though molecular dynamics simulations show that ShK is more flexible than HmK. Binding of the anti-KV1.3 nanobody A0194009G09 to the turret and residues in the external loops of the voltage-sensing domain enhances the dilation of the outer selectivity filter in an exaggerated inactivated conformation. These studies lay the foundation to further define the mechanism of slow inactivation in KV channels and can help guide the development of future KV1.3-targeted immuno-therapeutics.
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Simulación de Dinámica Molecular , Canales de Potasio con Entrada de Voltaje , Animales , Ratas , Transporte Biológico , Línea Celular , Conformación MolecularRESUMEN
Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.
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Dapsona , Células Asesinas Naturales , Perforina/metabolismo , Ligandos , Células Asesinas Naturales/metabolismo , Muerte Celular , Dapsona/metabolismoRESUMEN
Increased expression of the voltage-gated potassium channel Kv1.3 in activated microglia, and the subsequent release of pro-inflammatory mediators, are closely associated with the progression of Alzheimer's disease (AD). Studies have shown that reducing neuroinflammation through the non-selective blockade of microglial Kv1.3 has the potential to improve cognitive function in mouse models of familial AD. We have previously demonstrated that a potent and highly-selective peptide blocker of Kv1.3, HsTX1[R14A], not only entered the brain parenchyma after peripheral administration in a lipopolysaccharide (LPS)-induced mouse model of inflammation, but also significantly reduced pro-inflammatory mediator release from activated microglia. In this study, we show that microglial expression of Kv1.3 is increased in senescence accelerated mice (SAMP8), an animal model of sporadic AD, and that subcutaneous dosing of HsTX1[R14A] (1 mg/kg) every other day for 8 weeks provided a robust improvement in cognitive deficits in SAMP8 mice. The effect of HsTX1[R14A] on the whole brain was assessed using transcriptomics, which revealed that the expression of genes associated with inflammation, neuron differentiation, synapse function, learning and memory were altered by HsTX1[R14A] treatment. Further study is required to investigate whether these changes are downstream effects of microglial Kv1.3 blockade or a result of alternative mechanisms, including any potential effect of Kv1.3 blockade on other brain cell types. Nonetheless, these results collectively demonstrate the cognitive benefits of Kv1.3 blockade with HsTX1[R14A] in a mouse model of sporadic AD, demonstrating its potential as a therapeutic candidate for this neurodegenerative disease.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Ratones , Animales , Péptidos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Modelos Animales de Enfermedad , Inflamación , CogniciónRESUMEN
BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.
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Anémonas de Mar , Animales , Anémonas de Mar/genética , Genómica , Inversión Cromosómica , Cisteína , DisulfurosRESUMEN
Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.
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Venenos de Cnidarios , Anémonas de Mar , Animales , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/metabolismo , Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/farmacología , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Péptidos/química , Conformación Molecular , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/química , Canal de Potasio Kv.1.2/metabolismoRESUMEN
Phylum Cnidaria represents a unique group among venomous taxa, with its delivery system organised as individual organelles, known as nematocysts, heterogeneously distributed across morphological structures rather than packaged as a specialised organ. Acontia are packed with large nematocysts that are expelled from sea anemones during aggressive encounters with predatory species and are found in a limited number of species in the superfamily Metridioidea. Little is known about this specialised structure other than the commonly accepted hypothesis of its role in defence and a rudimentary understanding of its toxin content and activity. This study utilised previously published transcriptomic data and new proteomic analyses to expand this knowledge by identifying the venom profile of acontia in Calliactis polypus. Using mass spectrometry, we found limited toxin diversity in the proteome of acontia, with an abundance of a sodium channel toxin type I, and a novel toxin with two ShK-like domains. Additionally, genomic evidence suggests that the proposed novel toxin is ubiquitous across sea anemone lineages. Overall, the venom profile of acontia in Calliactis polypus and the novel toxin identified here provide the basis for future research to define the function of acontial toxins in sea anemones.
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Venenos de Cnidarios , Anémonas de Mar , Animales , Anémonas de Mar/química , Ponzoñas , Proteómica , Perfilación de la Expresión Génica , Nematocisto , Venenos de Cnidarios/genética , Venenos de Cnidarios/químicaRESUMEN
Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.
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Conotoxinas , Caracol Conus , Receptores Nicotínicos , Ratones , Humanos , Animales , Conotoxinas/farmacología , Caracol Conus/metabolismo , Ponzoñas , Receptores Nicotínicos/metabolismo , Péptidos/metabolismo , Antagonistas Nicotínicos/farmacologíaRESUMEN
The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 â¼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 â¼ 6 µM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.
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Lipopolisacáridos , Enfermedades Neuroinflamatorias , Ratones , Animales , Distribución Tisular , Radioisótopos de Galio/metabolismo , Ratones Endogámicos C57BL , Péptidos/química , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Inflamación/metabolismo , Tomografía de Emisión de PositronesRESUMEN
Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 â¼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.
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Canal de Potasio Kv1.3 , Venenos de Escorpión , Ratones , Animales , Cricetinae , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Cricetulus , Distribución Tisular , Péptidos/químicaRESUMEN
The ability to predict cell-permeable candidate molecules has great potential to assist drug discovery projects. Large molecules that lie beyond the Rule of Five (bRo5) are increasingly important as drug candidates and tool molecules for chemical biology. However, such large molecules usually do not cross cell membranes and cannot access intracellular targets or be developed as orally bioavailable drugs. Here, we describe a random forest (RF) machine learning model for the prediction of passive membrane permeation rates developed using a set of over 1000 bRo5 macrocyclic compounds. The model is based on easily calculated chemical features/descriptors as independent variables. Our random forest (RF) model substantially outperforms a multiple linear regression model based on the same features and achieves better performance metrics than previously reported models using the same underlying data. These features include: (1) polar surface area in water, (2) the octanol-water partitioning coefficient, (3) the number of hydrogen-bond donors, (4) the sum of the topological distances between nitrogen atoms, (5) the sum of the topological distances between nitrogen and oxygen atoms, and (6) the multiple molecular path count of order 2. The last three features represent molecular flexibility, the ability of the molecule to adopt different conformations in the aqueous and membrane interior phases, and the molecular "chameleonicity." Guided by the model, we propose design guidelines for membrane-permeating macrocycles. It is anticipated that this model will be useful in guiding the design of large, bioactive molecules for medicinal chemistry and chemical biology applications.
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Compuestos Macrocíclicos , Hidrógeno , Aprendizaje Automático , Nitrógeno , Octanoles , Oxígeno , AguaRESUMEN
Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.
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Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina , Proteínas Intrínsecamente Desordenadas , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Péptido Hidrolasas/metabolismo , Unión ProteicaRESUMEN
The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.
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Péptidos de Penetración Celular , Óxido Nítrico , Péptidos de Penetración Celular/farmacología , Macrófagos/metabolismo , Modelos Moleculares , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.
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Venenos de Hormiga/química , Hormigas/fisiología , Hipersensibilidad a las Drogas , Factor de Crecimiento Epidérmico/química , Toxinas Biológicas/química , Secuencia de Aminoácidos , Animales , Mordeduras y Picaduras de Insectos , Ratones , Imitación MolecularRESUMEN
Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.