Snake venom disintegrins update: insights about new findings

ABSTRACT Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use

Structural basis for pure antagonism of integrin αVβ3 by a high affinity form of fibronectin

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand: the 10th type III RGD-domain of wild-type fibronectin (wtFN10), or to a high affinity mutant (hFN10) that acts as a pure antagonist. Comparison of these structures revealed a central π - π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3-subunit that blocked conformational changes triggered by wtFN10, and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side-chains, or reorienting Trp1496 away from Tyr122, converted hFN10 into a partial agonist. The findings offer new insights on the mechanism of integrin activation and a basis for design of RGD-based pure antagonists

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.Fil: Mészáros, Bálint. European Molecular Biology Laboratory; AlemaniaFil: Sámano Sánchez, Hugo. European Molecular Biology Laboratory; AlemaniaFil: Alvarado Valverde, Jesús. European Molecular Biology Laboratory; Alemania. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Čalyševa, Jelena. European Molecular Biology Laboratory; Alemania. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Martinez Perez, Elizabeth. Fundación Instituto Leloir; Argentina. European Molecular Biology Laboratory; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alves, Renato. European Molecular Biology Laboratory; AlemaniaFil: Shields, Denis C.. Universidad de Dublin; IrlandaFil: Kumar, Manjeet. European Molecular Biology Laboratory; AlemaniaFil: Rippmann, Friedrich. Computational Chemistry & Biology; AlemaniaFil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Gibson, Toby James. European Molecular Biology Laboratory; Alemani

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs

Snake Venom

Venomous snakes belonging to the family Viperidae, Elapidae, Colubridae and Hydrophidae, produces snake venom in order to facilitate immobilization and digestion of prey, act as defense mechanism against threats. Venom contains zootoxins which is a highly modified saliva that is either injected via fangs during a bite or spitted. The modified parotid gland, encapsulated in a muscular sheath, present on each side of the head, below and behind the eye, have large alveoli which temporarily stores the secreted venom and later conveyed by a duct to tubular fangs through which venom is injected. Venoms are complex mixtures of more than 20 different compounds, mostly proteins and polypeptides, including proteins, enzymes and substances with lethal toxicity which are either neurotoxic or haemotoxic in action and exert effects on nervous/muscular impulses and blood components. Lots of research are directed to use venoms as important pharmacological molecules for treating various diseases like Alzheimer’s disease, Parkinson’s disease etc

Análise de arquétipos estruturais de desintegrinas para o desenvolvimento de moléculas inibidoras da agregação plaquetária

Dissertação (mestrado)—Universidade de Brasília, Instituto de Biologia, Programa de Pós Graduação em Biologia Animal, 2014.Os anfíbios anuros são animais que vêm sendo estudados e utilizados como modelos para descoberta de novas moléculas há algum tempo. Sua pele secreta uma mistura de substâncias ativas em grande parte já isoladas e estruturalmente caracterizadas. Dentre tais compostos, podemos citar peptídeos antimicrobianos, hipotensores e opióides, aminas biogênicas, alcaloides e esteróis. O trabalho em questão analisou um peptídeo oriundo da secreção cutânea do anfíbio Hypsiboas punctatus. Tal peptídeo apresenta uma sequência de 15 resíduos de aminoácidos e um motivo ativo composto pelo tripeptídeo KGD característico das proteínas da família das desintegrinas, até o momento inédito em anfíbios. O peptídeo em questão foi modificado em mais cinco análogos e sintetizado por meio de síntese química sólida. As moléculas foram purificadas, caracterizadas e testadas e, ao final do processo, tiveram os modelos das suas estruturas tridimensionais determinados por meio de Ressonância Magnética Nuclear. A sequência peptídica original foi modificada para gerar cinco análogos sintéticos obtidos por meio de síntese química em fase sólida. Todos os análogos foram submetidos à oxidação do par de resíduos de cisteína para formação das respectivas pontes dissulfeto, purificados, caracterizados e testados. Ao final deste processo, tiveram suas respectivas estruturas tridimensionais determinadas por meio de Ressonância Magnética Nuclear. O peptídeo natural não apresentou qualquer atividade anti- plaquetária in vitro quando testado por meio da metodologia de impedância elétrica, no entanto é possível atribuir-se a esta molécula discreta atividade relativa de inibição de agregação em ensaios de espalhamento plaquetário. Contudo, quando avaliados comparativamente sob mesma metodologia, os análogos sintéticos demonstram atividade anti-plaquetária significativamente superior ao peptídeo natural, utilizado neste trabalho como um arquétipo estrutural de desintegrina. Tais evidências, isto é, a comprovação experimental do surgimento de uma nova atividade biológica em análogos sintéticos, originalmente inexistente na molécula arquetípica, corrobora nossa hipótese sobre a importância da redistribuição das cargas (positiva e negativa) ao longo do eixo maior da estrutura 3D de cada análogo e da necessidade de um reposicionamento “Taylor-Made” dos motivos (Arg-Gly-Asp; Lys-Gly-Asp; His-Gly-Asp) para que as novas moléculas fossem ativas. ______________________________________________________________________________ ABSTRACTAnurans are animals that have been much explored as models. Their skin is part of their immune system and it has special glands which secrete a rich mixture of substances related to their innate defense against predators and microorganisms. Among this molecules are active peptides which have been isolated and tested for many pathogens and are a growing field within pharmacology. The present work analyzed a specific peptide from the skin secretion of the amphibian Hypsiboas punctatus. It is formed by a 15 amino acid sequence with a KGD disintegrin-like motif in the center of a loop constricted by a disulfide bridge. This motif has never been registered for amphibians before. The natural peptide was modified into five analogues which were synthesized through solid phase tButyl/FMOC strategy. They were purified (Reverse-Phase High Performance Liquid Chromatography), characterized (Mass Spectrometry), had their biological activity tested and their 3D structures calculated with the use of Nuclear Magnetic Resonance. The natural peptide did not show any anti-platelet inhibition activity when tested through electric impedance methodology; however it did show relative biological activity when tested through platelet spreading assay methodology. The modified analogues showed a higher activity rate. The results indicate the verification of the hypothesis of the importance of an improved positioning of the positive and negative charges along the molecule structure and also of the need for the ligand section of the peptide to be centered in the active loop

Proteomic Analyses of Snake Venoms with an Examination of the Biological Roles and Anti-cancer Effects of Venom Disintegrins

For decades, snakes and snake venoms have been utilized in numerous aspects of biological and biomedical research. Behaviorally, snakes have been examined for their extraordinary chemosensory capabilities, providing a detailed understanding of their foraging ecology and predatory responses. The presence of a highly complex vomeronasal organ has enabled snakes to not only respond to, but also discriminate between a high-range of heterospecific, conspecific, predatory, and prey-derived chemical odors. Snake venom has allowed for a transition in predatory behaviors, and this often complex mixture of proteins and peptides has provided researchers with an ever growing catalog of natural compounds that may be applicable as novel therapeutics or as biomedical reagents. Research into venomous systems also provides a detailed understanding of the biological roles of venom compounds, as well as providing critical information necessary for the proper assessment and treatment of snakebite. The current work addresses several aspects of snake behavior and snake venom toxinology and has four major objectives: i) to examine the chemosensory responses of neonate, subadult and adult Prairie Rattlesnake (Crotalus viridis viridis) to various prey chemical stimuli, ii) to identify the chemical component(s) of venom which allows for prey recovery during viperid predatory episodes, iii) to examine the anti-cancer effects of a novel snake venom disintegrin towards various human derived cancer cell lines and iv) to complete in-depth proteomic analyses of the neonate and adult C. v. viridis and examine the efficacy of the current anti-venom treatment CroFab® against this species’ venom. Chapter I presents the objectives and aims of my dissertation work, and provides background on chemosensory systems in squamates, and the numerous studies examining prey relocation in viperid snakes. Further, this chapter addresses the importance of examining the potential medicinal values of disintegrins as anti-cancer therapeutics, and the utilization of proteomics to develop a better understanding of venom composition and anti-venom efficacy. Chapters II focuses on the chemosensory responses of wild-caught neonate, subadult, and adult C. v. viridis to natural and non-natural prey-derived chemical odors. Results indicate that responses to chemical stimuli shift with snake age, correlating with ontogenetic changes in snake diet. Chapter III examines this phenomenon in more detail with a group of “stunted” C. v. viridis which had been in captivity since birth and had only consumed neonate lab mice (Mus musculus). Further, these snakes were the age of adults yet only the size of large juveniles, therefore they could not consume larger prey normally taken by adult snakes. Results suggest that ontogenetic shifts in responsiveness to natural prey chemical cues are innately programmed and are not based on body size or feeding experience. Chapter IV identifies the venom component, disintegrins, which are responsible for prey recovery during strike-induced chemosensory searching in Western Diamondback Rattlesnakes (Crotalus atrox). In Chapter V, a novel disintegrin protein (named tzabcanin) was isolated from the venom of the Middle American Rattlesnake (Crotalus simus tzabcan) and the cytotoxic and anti-adhesion properties of this protein toward Colo-205 and MCF-7 cell lines was examined. Chapter VI also examines the anticancer effects of tzabcanin towards A-375 and A-549 cell lines, and by specifically binding integrin αvβ3, tzabcanin inhibits cell migration and cell adhesion to vitronectin. In Chapter VII, a detailed proteomic analysis of the venoms of four individual C. v. viridis is presented, showing a novel trend in ontogenetic changes in venom composition, as well as identifying which compounds are, and which are not, effectively immunocaptured by the current anti-venom therapy used in the United States, CroFab®

The effect of two novel C-type lectins, Ba100 and Ba25, isolated from the venom of the puff adder, Bitis arietans on T lymphocyte proliferative responses

Includes abstract. Includes bibliographical references (p. 233-318)