Snake and arthropod venoms: search for inflammatory activity in human cells involved in joint diseases

Most anti-inflammatory drugs currently adopted to treat chronic inflammatory joint diseases can alleviate symptoms but they do not lead to remission. Therefore, new and more efficient drugs are needed to block the course of joint inflammatory diseases. Animal venoms, rich in bioactive compounds, can contribute as valuable tools in this field of research. In this study, we first demonstrate the direct action of venoms on cells that constitute the articular joints. We established a platform consisting of cell-based assays to evaluate the release of cytokines (IL-6, IL-8, TNFα, IL-1β, and IL-10) by human chondrocytes, synoviocytes and THP1 macrophages, as well as the release of neuropeptides (substance-P and β-endorphin) by differentiated sensory neuron-like cells, 24 h after stimulation of cells with 21 animal venoms from snake and arthropod species, sourced from different taxonomic families and geographic origins. Results demonstrated that at non-cytotoxic concentrations, the venoms activate at varying degrees the secretion of inflammatory mediators involved in the pathology of articular diseases, such as IL-6, IL-8, and TNF-α by chondrocytes, synoviocytes, and macrophages and of substance P by neuron-like cells. Venoms of the Viperidae snake family were more inflammatory than those of the Elapidae family, while venoms of Arthropods were less inflammatory than snake venoms. Notably, some venoms also induced the release of the anti-inflammatory IL-10 by macrophages. However, the scorpion Buthus occitanus venom induced the release of IL-10 without increasing the release of inflammatory cytokines by macrophages. Since the cell types used in the experiments are crucial elements in joint inflammatory processes, the results of this work may guide future research on the activation of receptors and inflammatory signaling pathways by selected venoms in these particular cells, aiming at discovering new targets for therapeutic intervention.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Proteomic analysis identifies molecular players and biological processes specific to SARS-CoV-2 exposure in endothelial cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

A Kunitz-type inhibitor from tick salivary glands: a promising novel antitumor drug candidate

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Losac and lopap recombinant proteins from Lonomia obliqua bristles positively modulate the myoblast proliferation process

The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE2) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE2 release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE2 in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE2, which is induced by an inflammatory stimulus by IL-1β. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.

Proliferation and invasion of melanoma are suppressed by a plant protease inhibitor, leading to downregulation of survival/death-related proteins

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

The phytoactive constituents of Eugenia selloi B.D. Jacks (pitangatuba): toxicity and elucidation of their anti-inflammatory mechanism(s) of action

We determined the phytochemical composition, anti-inflammatory mechanism of action, ROS/RNS scavenging capacity and systemic toxicity of a purified subfraction (S8) of Eugenia selloi. The composition of S8 was assessed by LC-ESI-QTOF-MS; the anti-inflammatory activity in RAW264.7 macrophages through NF-κB activation and biomarkers by multiplex in THP-1 cells; neutrophil migration, intravital microscopy and ICAM-1 expression in mice; NETs formation and CD11b expression; S8 scavenging capacity of ROS/RNS; toxicity in Galleria mellonella larvae model. Coumaric acid, quercetrin and vanillic acid were identified. S8 decreased NF-κB activation, IL-1β, IL-6, IL-10, MDC and MCP-1 levels, reduced neutrophil migration and ICAM-1 expression in mice; S8 did not interfere NET formation and CD11b expression, exhibited high antioxidant and showed negligible toxicity. E. selloi proved to be a promising, yet underexplored source of bioactive compounds, which can be useful employed in agribusiness and in the pharmaceutical and food industry to develop new products or human health supplies.

Lonomia obliqua envenoming and innovative research / Lonomia obliqua envenenamento e pesquisa Inovadora

As a tribute to Butantan Institute in its 120th anniversary, this review describes some of the scientific research efforts carried out in the study of Lonomia envenoming in Brazil, a country where accidents with caterpillars reach over 42,000 individuals per year (especially in South and Southeast Brazil). Thus, the promising data regarding the studies with Lonomia’s toxins contributed to the creation of new research centers specialized in toxinology based at Butantan Institute, as well as to the production of the antilonomic serum (ALS), actions which are in line with the Butantan Institute mission “to research, develop, manufacture, and provide products and services for the health of the population”. In addition, the study of the components of the Lonomia obliqua bristle extract led to the discovery of new molecules with peculiar properties, opening a field of knowledge that could lead to the development and innovation of new drugs aimed at cell regeneration and inflammatory diseases.

Lonomia obliqua venom induces NF-κB activation and a pro-inflammatory profile in THP-1-derived macrophage

Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars.

EcTI impairs survival and proliferation pathways in triple-negative breast cancer by modulating cell-glycosaminoglycans and inflammatory cytokines

Breast cancer is the most common malignant tumor among women worldwide, and triple-negative breast cancer is the most aggressive type of breast cancer, which does not respond to hormonal therapies. The protease inhibitor, EcTI, extracted from seeds of Enterolobium contortisiliquum, acts on the main signaling pathways of the MDA-MB-231 triple-negative breast cancer cells. This inhibitor, when bound to collagen I of the extracellular matrix, triggers a series of pathways capable of decreasing the viability, adhesion, migration, and invasion of these cells. This inhibitor can interfere in the cell cycle process through the main signaling pathways such as the adhesion, Integrin/FAK/SRC, Akt, ERK, and the cell death pathway BAX and BCL-2. It also acts by reducing the main inflammatory cytokines such as TGF-α, IL-6, IL-8, and MCP-1, besides NFκB, a transcription factor, responsible for the aggressive and metastatic characteristics of this type of tumor. Thus, the inhibitor was able to reduce the main processes of carcinogenesis of this type of cancer.

Losac, the first hemolin that exhibits procogulant activity through selective factor X proteolytic activation

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not ã-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X.

Losac, a factor X activator from Lonomia obliqua bristle extract Its role in the pathophysiological mechanisms and cell survival

Contact with the bristles of the caterpillar Lonomia obliqua can cause serious hemorrhage. Previously it was reported that a procoagulant protein (Lopap) in the bristle extract of L. obliqua increases cell longevity by inhibiting apoptosis. In this work, we purified from bristle extract a factor X activator that stimulates proliferation of endothelial cells. This protein, named Losac, was purified by ion exchange chromatography, followed by gel filtration chromatography and reverse-phase HPLC. Losac is a 45-kDa protein that activates factor X in a concentration-dependent manner and does not depend on calcium ions. In cultures of HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. HUVECs incubated with Losac (0.58 ìM for 1 h) increased release of nitric oxide and tissue-plasminogen activator, which both may mediate anti-apoptosis. Losac also increased slightly the decay-accelerating factor (DAF = CD55), which protects cells from complement-mediated lysis. On the other hand, Losac did not alter the release or expression of von Willebrand factor, tissue factor, intercellular adhesion molecule-1, interleukin-8, and prostacyclin. These characteristics indicate that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. Losac may have biotechnological applications, including the reduction of cell death and consequently increased productivity of animal cell cultures, and the use of hemolymph of L. obliqua for this purpose is already being explored. Further study is required to elucidate the mechanism for the inhibition of apoptosis by Losac.

Exploring new molecules and activities from Lonomia obliqua caterpillars

Skin contact with Lonomia caterpillar bristles causes a consumptive coagulopathy. From a cDNA library we cloned and expressed a prothrombin activator (rLopap) in active form, and from the bristles extract we characterized a FX activator (Losac). Several clones were sequenced and analyzed by expressed sequence tags. A database of about 1,270 sequences was constructed and deposited in NCBI. Both the native protein from the venom (Lopap) and the recombinant form (r-Lopap) promoted prothrombin hydrolysis, generating prethrombin-2, F1.2 and thrombin. Losac is a single-chain (43 kDa) protein that cleaves the FX heavy chain producing FXaá. In HUVECs rLopap and Losac are able to modulate cell survival by preventing apoptosis. rLopap increases NO and PGI2 concentration and Losac induces t-PA expression. Finally, to identify the venom proteins related to human envenomation, a 2D electrophoresis map is being performed as an attempt to find the majortoxins recognized by the anti-lonomia venom.

A prothrombin activator (Lopap) modulating inflammation, coagulation and cell survival mechanisms.

A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death.