RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential

Isolation and structural and functional characterization of Ts15, a new neurotoxin from the venom of the scorpion Tityus serrulatus

Os escorpiões são um dos grupos de animais mais antigos da Terra. Eles são artrópodes e pertecem a classe Arachinida e Ordem Scorpionida. A família Buthidae compreende as espécies responsáveis pelos acidentes graves em humanos, incluindo a espécie Tityus serrulatus, o maior responsável por esses acidentes no Brasil. A peçonha do T. serrulatus contém diversas neurotoxinas que agem especificamente em canais para sódio, potássio e cálcio da membrana plasmática de células excitáveis, causando massiva liberação de neurotransmissores.As toxinas escorpiônicas podem ser usadas como ferramentas nos estudos de estrutura e função desses canais iônicos sensíveis a voltagem e também no estudo de liberação e captação de neurotransmissores. As toxinas escôrpionicas específicas para canais para sódio sensíveis a voltagem são as principais responsáveis pelos efeitos do envenenamento por estes artrópodes e podem ser classificadas em duas classes: toxinas e . As -toxinas retardam a inativação desses canais induzindo assim um prolongamento na fase de repolarização do potencial de ação. As - toxinas alteram a dependência de voltagem de ativação dos canais para sódio para potenciais mais negativos provocando potenciais de ação espontâneos e repetitivos. As toxinas específicas para canais para potássio (KTx) são geralmente peptídeos pequenos e de caráter básico, formados por 23-43 aminoácidos estabilizados por 3-4 pontes dissulfeto. As KTx são classificadas em 4 subfamílias:, , , . Neste trabalho, uma nova neurotoxina do escorpião T. serrulatus foi isolada e caracterizada bioquímica e funcionalmente. A toxina foi testada em ampla variedade de canais incluindo 5 subtipos de canais para sódio (Nav1.4; Nav1.5; Nav1.6; Nav1.8 e DmNav1) e 12 diferentes tipos de canais para potássio (Kv1.1 a Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR e hERG). A peçonha bruta solúvel foi fracionada em cromatografia de troca iônica em coluna CM-Celulose-52 (2,5 cm x 63 cm), previamente equilibrada e eluída com tampão NH4HCO3 (pH 7,8). Essa primeira etapa cromatográfica permitiu a separação de 13 frações nomeadas de I XIII. A fração X foi submetida à cromatografia de fase reversa em sistema de cromatografia líquida de alta eficiência em que a toxina pura Ts15 pode ser obtida. Seu sequenciamento amino-terminal demonstrou que esse peptídeo possui 36 resíduos de aminoácidos estabilizados por 3 pontes dissulfeto. A massa molecular obtida por espectrometria de massa foi de 3956 e o pI predito pelo programa ProtParam foi de 8,86, no entanto, o pI determinado por focalização isolelétrica foi maior que 9,3. Os experimentos de eletrofisiologia utilizando as técnicas patch clamp e two microelectrode voltage clamp mostraram que a toxina Ts15 bloqueia preferencialmente os subtipos de canais para potássio Kv1.2 e Kv1.3 com IC50 de 196 ± 25 nM e 508± 67 nM respectivamente. Os ensaios de captação de neurotransmissores em sinaptosomas de cérebro de rato foram realizados adicionando 3H-GABA e 3H-Glu na presença e ausência de diferentes concentrações da toxina Ts15. Não foram observados efeitos nos canais para sódio em todas as concentrações testadas assim como na captação do GABA. Porém, foi observado aumento significante na captação do glutamato em todas as concentrações testadas, provavelmente como resultado de efeito secundário da ação da Ts15 em canais para potássio sensível a voltagem. Em conclusão, a Ts15 pode ser considerada um autêntico novo tipo de toxina escorpiônica, com afinidade para canais para potássio Kv1.2 e Kv1.3 e capaz de aumentar a captação de glutamato. Essa toxina é o único membro da nova subfamília -Ktx21 e portanto nomeada -Ktx21.1Scorpions are one of the most ancient groups of animals on earth. They are arthropods and belong to the class Arachinida and Order Scorpionida. The Buthidae family comprises the species that are really dangerous for human, including Tityus serrulatus that is responsible for most severe accidents in Brazil. T. serrulatus venom contains several neurotoxins that specifically act on sodium, potassium or calcium channels in excitable membranes, causing a massive release of neurotransmitters and leading to the stimulation of the autonomic nervous system. Since ion channels play important roles in many physiological processes, scorpion toxins have been used as tools for studies of the neurophysiological mechanisms involving voltage-gated ion channels and neurotransmitter release/uptake. Voltage-gated Na+ channel (Nav channel) toxins are mainly responsible of the harmful effects of scorpion venom and can be classified into two classes: and -neurotoxins. The -toxins retard Nav channel inactivation and induce a prolongation of the repolarization phase of the action potential. The -toxins shift the voltage dependence of Nav channel activation to more negative potentials that result in an increased tendency of the cell to fire spontaneously and repetitively. Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: , , and . These peptides display varying selectivity and affinity for different Kv channel subtypes. In this work, a novel toxin from the T. serrulatus venom was isolated, biochemistry and pharmacologically characterized using a wide electrophysiological screening on 5 different subtypes of Nav channels (Nav1.4; Nav1.5; Nav1.6; Nav1.8 and DmNav1) and 12 different subtypes of Kv channels (Kv1.1 - Kv1.6; Kv2.1; Kv3.1; Kv4.2; Kv4.3; Shaker IR and hERG). The crude soluble T. serrulatus venom was fractionated by ion exchange chromatography on a CM-cellulose-52 column (2.5 cm x 63.0 cm), which was equilibrated and eluted with NH4HCO3 buffer (pH 7.8). This chromatography allowed the separation of 13 fractions which were named I to XIII. Fraction X was submitted to a reverse-phase C18 (0.46 cm x 25 cm) high performance liquid chromatography (RP-HPLC) and the pure toxin, Ts15, could be obtained. The amino acid sequence of this novel peptide showed that it contains 36 amino acids and is cross-linked by 3 disulfide bridges. The molecular mass of Ts15 (3956) was obtained by electrospray (ESI) triple-quadrupole mass spectrometry and its pI value (8,86) was predicted by ProtParam program. However, the pI determined by isoeletric focusing was greater than 9,3. Electrophysiological experiments using patch clamp and the two electrode voltage clamp technique, showed that Ts15 preferentially blocks Kv1.2 and Kv1.3 channels with IC50 value of 196 ± 25 and 508 ± 67 nM, respectively. Uptake assays were performed by adding 3H-GABA and 3H-Glu, in the absence (controls) or presence of different concentrations of Ts15, on isolated rat brain synaptosomes. No effect on Nav channels was observed, in all tested concentrations, as well as for GABA uptake. However, Ts15 induced a significant increase of the glutamate uptake, probably as a secondary effect of its action on Kv channels. In conclusion, Ts15 can be considered a bonafide novel type of scorpion toxin that presents high affinity by Kv1.2 and Kv1.3 channels and was able to increase the glutamate uptake. It is the unique member of the new -Ktx21 subfamily and therefore was named -Ktx21.

Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors

peer reviewedaudience: researcher, professional, studentG-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF–TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCR

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Abstract Background: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology

Proteomic landscapes of Pachycondila villosa ant venom by nano-scale chromatography and high resolution mass spectrometry

Introduction: It is estimated that the total number of molecules present in animal venoms is a collection of 40 million different compounds and, despite the efforts made, less than 0,01% of those compounds was identified and characterized to date. However, recent progresses in proteomic, in parallel with the advances of mass spectrometry have contributed to the study of those bio-libraries. The sensitivity improvement of these instruments allows the study of minimal amounts of sample still yielding a wealth of information. The present work aimed to perform a deep proteomic analysis of the venom from the ant Pachycondilyla villosa focusing on the de novo sequencing and the characterization of post translational modifications using high resolution mass spectrometers. Methods: The crude venom (0,5 ug) of P. villosa ants collected on Panga Natural Reserve (Uberlandia-Minas Gerais- Brazil) was diluted in 0,2% of formic acid and injected into a nanoACQUITY ULPC equipped with a monolithic PepSwift Capillary column 100µm x 25, hyphened to a Q Exactive Orbitrap mass spectrometer. The elution of the compounds was performed with a gradient of 3 to 50% of solution B in 80 minutes (A: H2O/FA 0.1%; B: ACN) at flow rate of 1 µL/min. All mass spectrometry analyses were performed in data dependent analysis (DDA) mode that automatically triggers the MS/MS experiments. The top 10 most intense peaks of each MS scan was fragmented by high-energy dissociation (HCD) and their corresponding MS/MS spectra were acquired. Preliminary data: Animal venoms are considered a rich source of biologically active compounds, which has been constantly selected and refined by the processes of natural evolution, in which each molecule is endowed with pharmacological properties highly valuable for scientific purposes. Despite the commitment, the exploration of these bio-libraries remains limited which might be related to the technological limitations that prevent full-scale investigation of these venoms. In addition, the conventional methods used to explore animal venoms are still time-consuming and require large amounts of samples, which restrict the studies for a few species. Unquestionably, the advances of proteomics and mass spectrometry instrumentations benefited a great deal the research on hymenoptera venom. Mostly due to their small size and therefore scarcely collected venom, this order has always been neglected and considered unfeasible to be studied through the known strategies. The present work represents the first report concerning the venom composition of P. villosa ant. The preliminary results already highlight the complexity of this venom, which showed to be composed by over 5000 different molecules. Most of those components fall into the 800- 4000 Da range, which is in agreement with other studies regarding ant´s venom composition. Most of the proteomics studies concerning ant venoms already revealed the presence of linear peptides below 5000 Da as major components. Those small peptides usually display antimicrobial activity and some of them hold additional insecticidal activity. Novel aspect: The results obtained already point out the biotechnological potential of P. villosa venom and highlight’s its complexit

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

<div><p>Abstract Background: Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 800–4000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family. In addition, smaller fragments related to ponericins were also identified, suggesting that this class of antimicrobial peptide might undergo enzymatic cleavages. Conclusion: There are substantial differences among the venom of N. villosa ants collected in different seasons and from different nest habitats. The venom composition is affected by climate changes that influence prey availability and predator presence. Clearly, nano-LC-MS boosted the knowledge about ant venom, a rich source of unexplored and promising bioactive compounds.</p></div

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Abstract Background Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads’ secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman’s degradation. Results We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored