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.

Detection of SARS-CoV-2 virus via dynamic light scattering using antibody-gold nanoparticle bioconjugates against viral spike protein.

Mass testing for the diagnosis of COVID-19 has been hampered in many countries owing to the high cost of genetic material detection. This study reports on a low-cost immunoassay for detecting SARS-CoV-2 within 30 min using dynamic light scattering (DLS). The immunosensor comprises 50-nm gold nanoparticles (AuNPs) functionalized with antibodies against SARS-CoV-2 spike glycoprotein, whose bioconjugation was confirmed using transmission electron microscopy (TEM), UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and surface-enhanced Raman scattering spectroscopy (SERS). The specific binding of the bioconjugates to the spike protein led to an increase in bioconjugate size, with a limit of detection (LOD) 5.29 × 103 TCID50/mL (Tissue Culture Infectious Dose). The immunosensor was also proven to be selective upon interaction with influenza viruses once no increase in size was observed after DLS measurement. The strategy proposed here aimed to use antibodies conjugated to AuNPs as a generic platform that can be extended to other detection principles, enabling technologies for low-cost mass testing for COVID-19.

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.

Detection of SARS-CoV-2 virus via dynamic light scattering using antibody-gold nanoparticle bioconjugates against viral spike protein

Mass testing for the diagnosis of COVID-19 has been hampered in many countries owing to the high cost of genetic material detection. This study reports on a low-cost immunoassay for detecting SARS-CoV-2 within 30 min using dynamic light scattering (DLS). The immunosensor comprises 50-nm gold nanoparticles (AuNPs) functionalized with antibodies against SARS-CoV-2 spike glycoprotein, whose bioconjugation was confirmed using transmission electron microscopy (TEM), UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and surface-enhanced Raman scattering spectroscopy (SERS). The specific binding of the bioconjugates to the spike protein led to an increase in bioconjugate size, with a limit of detection (LOD) 5.29 × 103 TCID50/mL (Tissue Culture Infectious Dose). The immunosensor was also proven to be selective upon interaction with influenza viruses once no increase in size was observed after DLS measurement. The strategy proposed here aimed to use antibodies conjugated to AuNPs as a generic platform that can be extended to other detection principles, enabling technologies for low-cost mass testing for COVID-19.

rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii.

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 µM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

rDromaserpin: a novel anti-hemostatic serpin, from the salivary glands of the hard tick Hyalomma dromedarii

Hemostatic disorders are caused either by platelet-related dysfunctions, defective blood coagulation, or by a combination of both, leading to an increased susceptibility to cardiovascular diseases (CVD) and other related illnesses. The unique specificity of anticoagulants from hematophagous arthropods, such as ticks, suggests that tick saliva holds great promise for discovering new treatments for these life-threatening diseases. In this study, we combined in silico and in vitro analyses to characterize the first recombinant serpin, herein called Dromaserpin, from the sialotranscriptome of the Hyalomma dromedarii tick. Our in silico data described Dromaserpin as a secreted protein of ~43 kDa with high similarities to previously characterized inhibitory serpins. The recombinant protein (rDromaserpin) was obtained as a well-structured monomer, which was tested using global blood coagulation and platelet aggregation assays. With this approach, we confirmed rDromaserpin anticoagulant activity as it significantly delayed plasma clotting in activated partial thromboplastin time and thrombin time assays. The profiling of proteolytic activity shows its capacity to inhibit thrombin in the micromolar range (0.2 to 1 μM) and in the presence of heparin this inhibition was clearly increased. It was also able to inhibit Kallikrein, FXIa and slightly FXIIa, with no significant effect on other factors. In addition, the rDromaserpin inhibited thrombin-induced platelet aggregation. Taken together, our data suggest that rDromaserpin deserves to be further investigated as a potential candidate for developing therapeutic compounds targeting disorders related to blood clotting and/or platelet aggregation.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Sulfate-Binding Protein (Sbp) from Xanthomonas citri: Structure and Functional Insights.

The uptake and transport of sulfate in bacteria is mediated by an ATP-binding cassette transporter (ABC transporter) encoded by sbpcysUWA genes, whose importance has been widely demonstrated due to their relevance in cysteine synthesis and bacterial growth. In Xanthomonas citri, the causative agent of canker disease, the expression of components from this ABC transporter and others related to uptake of organic sulfur sources has been shown during in vitro growth cultures. In this work, based on gene reporter and proteomics analyses, we showed the activation of the promoter that controls the sbpcysUWA operon in vitro and in vivo and the expression of sulfate-binding protein (Sbp), a periplasmic-binding protein, indicating that this protein plays an important function during growth and that the transport system is active during Citrus sinensis infection. To characterize Sbp, we solved its three-dimensional structure bound to sulfate at 1.14 Å resolution and performed biochemical and functional characterization. The results revealed that Sbp interacts with sulfate without structural changes, but the interaction induces a significant increasing of protein thermal stability. Altogether, the results presented in this study show the evidence of the functionality of the ABC transporter for sulfate in X. citri and its relevance during infection.

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases.

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.

Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain.

Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 A resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 A. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.

Interação Trypanosoma cruzi-célula hospedeira: estudo do "domínio FLY", motivo carboxi-subterminal conservado na superfamília das gp85/trans-sialidases / Interaction between Trypanosoma cruzi and host cell: study of FLY domain, a conserved carboxil-subterminal motiv of gp85/trans-sialidases

Trypanosoma cruzi, agente causador da Doença de Chagas, é um protozoário intracelular obrigatório. Vários estudos foram realizados visando a caracterização de moléculas de 85-90 kDa, presentes na superfície do parasita bem como de seus possíveis receptores nas células do hospedeiro vertebrado. Um membro da superfamília das gp85/trans-sialidases (Tc85-11), expresso somente na superfície das formas intectivas tripomastigotas e que adere em laminina e em células, foi clonado e caracterizado em nosso laboratório. Peptídeo J, fragmento de Tc85-11 não implicado em adesão a laminina, que contém o motivo conservado na superfamília, com seqüência VTVXNVFLYNR, aqui denominado "domínio FLY", foi identificado como sendo responsável pela adesão da porção carboxi-terminal da proteína em células epiteliais e, adicionado ao meio de cultura, promoveu aumento do número de células infectadas por T.cruzi. Seu receptor foi descrito como CK18 (Magdesian et al., 2001). Dando continuidade a esse estudo, em nosso laboratório caracterizamos a porção amino-terminal de CK18 como a região da interação com "domínio FLY" e identificamos o provável sítio de ligação, localizado entre os 15 aminoácidos iniciais da proteína. Adicionalmente, com o intuito de determinar a função do "domínio FLY", caracterizamos o peptídeo J como molécula extremamente adesiva, interagindo com a superfície de células epiteliais, matriz extracelular e promovendo interação entre tripomastigotas e ECM, possivelmente por meio de interações hidrofóbicas não dependentes de sua estrutura tridimensional adotada na proteína nativa. "Domínio FLY" foi caracterizado, ainda, como possível modulador da infecção por tripomastigotas já que, da mesma forma que estimula a invasão quando adicionado ao meio de cultura (Magdesian et al., 2001), promove secreção de proteínas imunorelacionadas com CK18 e ligantes de proteína A para o sobrenadante celular...