DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913University of Maryland Baltimore County Designated Research Initiative Fund, an Undergraduate Research AwardUniversity of Maryland Baltimore County Designated Research Initiative Fund, an Undergraduate Research AwardFundao de Amparo a Pesquisa do Estado de So Paulo [FAPESP]Fundao de Amparo a Pesquisa do Estado de So PauloNational Council of Technological and Scientific Development [CNPq]National Council of Technological and Scientific Developmen

State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources) has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP) with a more pronounced antifungal activity. in contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. in vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. the structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Inst Butantan, Genet Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, São Paulo, BrazilUniv Fed Ceara, Labomar Inst Ciencias Mar, Fortaleza, CE, BrazilUniv Estado Amazonas, Manaus, AM, BrazilCBA, Lab Bioquim & Biol Mol, Manaus, AM, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, São Paulo, BrazilWeb of Scienc

Disorders of sex development: effect of molecular diagnostics

Disorders of sex development (DSDs) are a diverse group of conditions that can be challenging to diagnose accurately using standard phenotypic and biochemical approaches. Obtaining a specific diagnosis can be important for identifying potentially life-threatening associated disorders, as well as providing information to guide parents in deciding on the most appropriate management for their child. Within the past 5 years, advances in molecular methodologies have helped to identify several novel causes of DSDs; molecular tests to aid diagnosis and genetic counselling have now been adopted into clinical practice. Occasionally, genetic profiling of embryos prior to implantation as an adjunct to assisted reproduction, prenatal diagnosis of at-risk pregnancies and confirmatory testing of positive results found during newborn biochemical screening are performed. Of the available genetic tests, the candidate gene approach is the most popular. New high-throughput DNA analysis could enable a genetic diagnosis to be made when the aetiology is unknown or many differential diagnoses are possible. Nonetheless, concerns exist about the use of genetic tests. For instance, a diagnosis is not always possible even using new molecular approaches (which can be worrying for the parents) and incidental information obtained during the test might cause anxiety. Careful selection of the genetic test indicated for each condition remains important for good clinical practice. The purpose of this Review is to describe advances in molecular biological techniques for diagnosing DSDs

Myotoxin a, crotamine and defensin homologs in reptile venoms

Abstract - Snakebite envenomation is common in all inhabited continents of the world, with more than 100,000 fatalities occurring every year. Depending on the snake species involved, envenomation includes often disabling intense local tissue damage with inflammation, pain and myonecrosis, caused in part by molecules known as myotoxins. Myotoxins are found in the venoms of several snakes, and their homologs are also found in lizards and mammals (the Platypus). These small peptide toxins show a unique structural/physicochemical resemblance to β-defensins, which are anti-microbial peptides (AMPs) involved in the resistance of epithelial surfaces to microbial colonization. These similarities suggest a possible common phylogenetic and/or functional association among these myotoxic and anti-microbial peptides. These β-defensins seems to play a key role as toxins in the envenomation process by activating the immune system. Crotamine, purified from the venom of the South American Rattlesnake Crotalus durissus terrificus, is one of the first myotoxins to be characterized. In addition to its role in rapid prey incapacitation, several therapeutic applications of myotoxin a like peptides (mainly crotamine), for instance, as antitumor and anti-microbial agents, have been characterized and are listed in this chapter. The several different characterized biological activities exhibited by these myotoxin-like peptides are mostly dependent on their three-dimensional structure and positively charged surface (cationic feature). Herein, we present the main known myotoxins of venoms, introduce some alternatives for neutralizing their effects in snakebite envenomations, and discuss their mechanism of action(s), designated biological functions, and structural/functional similarities with AMPs (β-defensins). This chapter will therefore discuss the structure, biological activities and differences/similarities shared between the toxins with antitumoral/anti-microbial activities (namely, myotoxin-a and crotamine) and the immune effector (defensin-like) polypeptides. Interestingly, toxins and immune effectors share similar evolutionary and structural patterns, and they both seem to have evolved to defend against the threats of potential predators, microbial invasions and/or malignant cells