South American rattlesnake cationic polypeptide crotamine trafficking dynamic in Plasmodium falciparum-infected erythrocytes: Pharmacological inhibitors, parasite cycle and incubation time influences in uptake.

Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.

Biophysical and biological properties of small linear peptides derived from crotamine, a cationic antimicrobial/antitumoral toxin with cell penetrating and cargo delivery abilities.

Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.

Gene expression alterations related to mania and psychosis in peripheral blood of patients with a first episode of psychosis.

Psychotic disorders affect ~3% of the general population and are among the most severe forms of mental diseases. In early stages of psychosis, clinical aspects may be difficult to distinguish from one another. Undifferentiated psychopathology at the first-episode of psychosis (FEP) highlights the need for biomarkers that can improve and refine differential diagnosis. We investigated gene expression differences between patients with FEP-schizophrenia spectrum (SCZ; N=53) or FEP-Mania (BD; N=16) and healthy controls (N=73). We also verified whether gene expression was correlated to severity of psychotic, manic, depressive symptoms and/or functional impairment. All participants were antipsychotic-naive. After the psychiatric interview, blood samples were collected and the expression of 12 psychotic-disorder-related genes was evaluated by quantitative PCR. AKT1 and DICER1 expression levels were higher in BD patients compared with that in SCZ patients and healthy controls, suggesting that expression of these genes is associated more specifically to manic features. Furthermore, MBP and NDEL1 expression levels were higher in SCZ and BD patients than in healthy controls, indicating that these genes are psychosis related (independent of diagnosis). No correlation was found between gene expression and severity of symptoms or functional impairment. Our findings suggest that genes related to neurodevelopment are altered in psychotic disorders, and some might support the differential diagnosis between schizophrenia and bipolar disorder, with a potential impact on the treatment of these disorders.

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Anthelmintic effects of a cationic toxin from a South American rattlesnake venom.

Despite the unquestionable importance of the highly cationic feature of several small polypeptides with high content of positively charged amino acids for their biological activities, positively charged peptides do not necessarily have the capacity to cross the cell membranes. Interestingly, we found that crotamine, a positively charged amphiphilic peptide from the South American rattlesnake venom, has a unique cell-penetrating property with affinity for acidic vesicles, besides a well-characterized antimicrobial and antitumoral activities. In spite of a remarkable in vitro antifungal activity of crotamine against Candida spp., no significant effect of this peptide could be observed in the course of Candida albicans and Candida krusei infection on Caenorhabditis elegans asssed in vivo. These experiments, in which the nematode C. elegans was used as a living host, suggested, however, the potential anthelmintic activity of crotamine because of its uptake by the worms and accumulation in their acidic compartments. As described in the present work, this lysosomotropic property is consistent with a previously proposed mechanism of toxicity of crotamine on mammalian tumoral cell lines. This study also allowed us to propose the cationic peptides with lysosomotropic property, as crotamine, as a potential new class of anthelmentics with ability to overcome the challenging problems of drug resistance.

Convergent evidences from human and animal studies implicate angiotensin I-converting enzyme activity in cognitive performance in schizophrenia.

In schizophrenia (SCZ), higher angiotensin I-converting enzyme (ACE) levels have been reported in patient's blood and cerebrospinal fluid (CSF). Hereby, we propose to explore whether the ACE activity levels are associated to cognitive performance in SCZ. Seventy-two patients with SCZ or schizoaffective disorder diagnosis, and 69 healthy controls (HCs) underwent a cognitive battery with parallel collection of peripheral blood samples to measure ACE activity. Significant higher ACE activity levels were confirmed in the plasma of SCZ patients compared with HCs (Student's t=-5.216; P<0.001). ACE activity significantly correlated to Hopkins delayed recall measures (r=-0.247; P=0.004) and Hopkins total (r=-0.214; P=0.012). Subjects grouped as high ACE activity (above average) had worse performance compared with low ACE activity level group for Hopkins delayed recall measure, even after correction for clinical condition, age, gender and years of education (P=0.029). The adjusted R squared for this final model was 0.343. This result was evident only comparing extreme groups for ACE activity, when splitting the sample in three groups with similar number of subjects. To clarify this finding, we performed an evaluation of the cognitive performance of transgenic mice with three copies of ACE gene in novel object recognition (NOR) test, which showed that such animals presented impairment in NOR (P<0.05) compared with two copies of wild-type animals. The results observed in SCZ patients and animal model suggest both the association of ACE to cognitive deficits in SCZ. This finding may support the evaluation of novel treatment protocols and/or of innovative drugs for specific intervention of cognitive deficits in SCZ envisioning concomitant ACE activity and behavior evaluations.

Protein-Protein and Peptide-Protein Interactions of NudE-Like 1 (Ndel1): A Protein Involved in Schizophrenia.

Schizophrenia (SCZ) is a devastating chronic mental disease determined by genetic and environmental factors, which susceptibility may involve an impaired neural migration during the neurodevelopmental process. Several candidate risk genes potentially associated with SCZ were related to the formation of protein complexes that ultimately mediate alterations in the neuroplasticity. The most studied SCZ risk gene is the Disrupted-in-Schizophrenia 1 (DISC1) gene, which functions seem to depend on the binding with cytoskeleton proteins, as the Nuclear-distribution gene E homolog like-1 (Ndel1) protein among others. Interestingly, Ndel1 is the only binding partner of DISC1 proteins with oligopeptidase activity, besides playing roles in multiple processes, including cytoskeletal organization, cell signaling, neuron migration, and neurite outgrowth. It is still not clear if the protein-protein interaction between Ndel1 and DISC1 is enough to explain all cellular functions attributed to these proteins, but there are several lines of evidence suggesting the importance of the catalytic activity of Ndel1 for the neurite outgrowth and neuron migration during embryogenesis. Recent works of the group have demonstrated the modulation of Ndel1 activity by DISC1, which is hypothetically impaired in SCZ patients. In fact, more recently, we also showed a lower Ndel1 activity in the plasma of SCZ patients compared to control health subjects, but the physiopathological significance of this feature is still unknown. Here we discuss Ndel1 ligands involved in protein-protein complex formations related to neurodevelopmental diseases, as (1) lissencephaly or Miller-Dieker Syndrome (MDS), which is characterized by the typical craniofacial features and abnormal smooth cerebral surface, and as (2) SCZ, since they both seem to be determined by defects in neuronal migration. Although impaired lissencephaly protein Lis1 complex formation with Ndel1 is the leading cause of lissencephaly, this binding does not affect Ndel1 oligopeptidase activity. On the other hand, although MDS and SCZ may be both determined by an abnormal neuronal migration, DISC1 complex formation with Ndel1 was shown to inhibit Ndel1 activity. Also differently of MDS, SCZ needs inputs from environmental factors, while lissencephaly is not likely dependent or affected by the environment. Several other proteins and peptide ligands were described for Ndel1, Lis1 and DISC1, thanks to the employment of biochemical, immunochemical, and biological (using cells or living animals) assays, including heterologous expression and also simply by purification from nature of these proteins in the complex form. Effects of the post-translational modifications of these proteins are also discussed here. Taken together, the data presented here show in essence how protein-protein and proteinpeptide interactions can underlie fundamental processes as cell division, maturation and migration, necessary for adequate formation of a complex structured tissue as the brain. A special attention was given to Ndel1 as this protein binds to either proteins or peptides, besides having proteolytic activity. Moreover, Ndel1 seems to be the key protein underlying two seemingly unrelated diseases with highly complex etiology, as lissencephaly and SCZ.

Gene expression analysis by ESTs sequencing of the Brazilian frog Phyllomedusa nordestina skin glands.

The subfamily Phyllomedusinae has attracted a great interest of many researchers mainly due to the high diversity of these frog species and plethora of pharmacological activities frequently observed for their skin secretions. Despite of this fact, mainly for new species, limited information is available regarding the molecular composition of these skin secretions and the cellular components involved in their production. Phyllomedusa nordestina is a recently described Brazilian frog species also popularly known as 'tree-frogs'. Aiming at contributing to the biological knowledge of this species, we show here the gene expression profile of this frog skin secretion using a global ESTs analysis of a cDNA library. The marked aspect of this analysis revealed a significant higher transcriptional level of the opioid peptide dermorphins in P. nordestina skin secretion than in Phyllomedusa hypochondrialis, which is its closest related species, belonging both to the same phylogenetic group. Precursors of bioactive peptides as dermaseptins, phylloseptins, tryptophyllins, and bradykinin-like peptideswere also found in this library. Transcripts encoding proteins related to ordinary cellular functions and pathways were also described. Some of them are chiefly involved in the production of the skin secretion. Taken together, the data reported here constitute a contribution to the characterization of the molecular diversity of gene-encoded polypeptides with potential possibility of pharmacological exploitation. The transcriptional composition of the skin secretion may also help to give the necessary support for the definition of P. nordestina as a new species, which actually relies basically on frog morphological characteristics and geographical distribution.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2and M1 muscarinic acetylcholine receptors.

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotesvasodilatation mediated by both bradykinin B2 and M1 muscarinicacetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venomglands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme(ACE) described. Bj-PRO-5a (

Identification of crotasin, a crotamine-related gene of Crotalus durissus terrificus.

Crotamine is a cationic peptide (4.9 kDa, pI 9.5) of South American rattlesnake, Crotalus durissus terrificus' venom. Its presence varies according to the subspecies or the geographical locality of a given species. At the genomic level, we observed the presence of 1.8 kb gene, Crt-p1, in crotamine-positive specimens and its absence in crotamine-negative ones. In this work, we described a crotamine-related 2.5 kb gene, crotasin (Cts-p2), isolated from crotamine-negative specimens. Reverse transcription coupled to polymerase chain reaction indicates that Cts-p2 is abundantly expressed in several snake tissues, but scarcely expressed in the venom gland. The genome of crotamine-positive specimen contains both Crt-p1 and Cts-p2 genes. The present data suggest that both crotamine and crotasin have evolved by duplication of a common ancestor gene, and the conservation of their three disulfide bonds indicates that they might adopt the same fold as beta-defensin. The physiological function of the crotasin is not yet known.

Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus)

A cDNA phage library was constructed from venom glands of a single adult specimen of crotamine-plus Crotalus durissus terrificus (South American rattlesnake) captured in a known region. Fifteen crotamine positive clones were isolated using a PCR-based screening protocol and sequenced. These complete cDNAs clones were grouped for maximal alignment into six distinct nucleotide sequences. The crotamine cDNAs, with 340-360 bases, encompass open reading frame of 198 nucleotides with 5' and 3' untranslated regions of variable size, signal peptide sequence, one crotamine isoform message, and putative poly(A+) signal. Of these six different crotamine cDNA precursors, two predict the identical amino acid sequence previously described by , and the other four a crotamine isoform precursor where the Leucine residue at position 19 is replaced by isoleucine by a single base change. On the other hand, nucleotide variation was observed in the 5' and 3' untranslated regions, with one interesting variant containing an 18 base pair deletion at the 5' untranslated region which results in the usual ATG initiator being replaced by the rarely used GUG start codon.Comparison by Northern blot analysis of poly(A+) RNA from venom glands of a crotamine-plus specimen to total and poly(A+) RNA from a crotamine-minus snake indicated that crotamine transcripts were not expressed in the crotamine-minus specimen.