Mirian A. F. Hayashi

Crotamine Mediates Gene Delivery into Cells through the Binding to Heparan Sulfate Proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme

The bradykinin‐potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin‐converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin‐converting enzyme in blood pressure regulation. In the present study, seven bradykinin‐potentiating peptides were identified within the C‐type natriuretic peptide precursor cloned from snake brain. The bradykinin‐potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin‐converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin‐potentiating peptides, one of which displays high specificity toward the N‐domain active site of the somatic angiotensin‐converting enzyme. In situ hybridization studies revealed the presence of the bradykinin‐potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin‐potentiating peptides, their presence within the neuroendocrine regulator C‐type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

Properties of cell penetrating peptides (CPPs)

Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell‐penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells. IUBMB Life, 58: 7 ‐ 13, 2006

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 in after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.

The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

Structural basis of the lisinopril-binding specificity in N- and C-domains of human somatic ACE.

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase which converts angiotensin I into the vasopressor peptide angiotensin II and also inactivates the hypotensive peptide bradykinin, playing an important role in blood pressure regulation. The present work describes the molecular modeling of the N-terminal human somatic ACE in complex with the inhibitor lisinopril, identifying the residues involved in the inhibitor-binding pocket. The obtained results identify differences in the lisinopril lysine moiety-binding residues for N- and C-terminals of sACE domains and an important carboxy-terminal proline hydrophobic accommodations mediated by the aromatic ring of Tyr532 and Tyr1128 residues, respectively. The present model will be useful for the development of a new inhibitor family based on the natural BPP peptides and derivatives, or even to improve the binding capacities and the domain specificity of the already known inhibitors.

Assessing the role of endooligopeptidase activity of Ndel1 (nuclear-distribution gene E homolog like-1) in neurite outgrowth

Ndel1 plays multiple roles in neuronal development but it is unknown whether its reported cysteine protease activity is important for these processes. Ndel1 is known to be critical for neurite outgrowth in PC12 cells where it works co-operatively in a complex with DISC1 to allow normal neuritogenesis. Through an initial interest in understanding the regulation of the expression of Ndel1 during neuronal differentiation, we have been able to show that Ndel1 expression and enzyme activity is up-regulated during neurite outgrowth in PC12 cells induced to neural differentiation. Heterologous expression of wild-type Ndel1 (Ndel1(WT)) in PC12 cells increases the percentage of cells bearing neurites in contrast to the catalytically dead mutant, Ndel1(C273A), which caused a decrease. Furthermore depletion of endogenous Ndel1 by RNAi decreased neurite outgrowth, which was rescued by transfection of the enzymatically active Ndel1(WT), but not by the Ndel1(C273A) mutant. Together these data support the notion that the endooligopeptidase activity of Ndel1 plays a crucial role in the differentiation process of PC12 cells to neurons. Genetic data and protein interaction with DISC1 might suggest a role for Ndel1 in neuropsychiatirc conditions.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline‐rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj‐PROs), are the first described naturally occurring inhibitors of the angiotensin I‐converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj‐PRO‐10c (

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.

Interaction of the rattlesnake toxin crotamine with model membranes.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. A common gene ancestry and structural similarity with the antimicrobial β-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against Candida spp., Trichosporon spp., and Cryptococcus neoformans. Crotamines selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several Candida spp. strains, except for a limited effect against C. krusei biofilm.