Antiplatelet mechanism of a subtilisin-like serine protease from Solanum tuberosum (StSBTc-3).

The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bß3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bß3 preventing the interaction between α2bß3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.

Bioconjugated liquid-like solid enhances characterization of solid tumor - chimeric antigen receptor T cell interactions.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable success as an immunotherapy for hematological malignancies, and its potential for treating solid tumors is an active area of research. However, limited trafficking and mobility of T cells within the tumor microenvironment (TME) present challenges for CAR T cell therapy in solid tumors. To gain a better understanding of CAR T cell function in solid tumors, we subjected CD70-specific CAR T cells to a challenge by evaluating their immune trafficking and infiltration through a confined 3D microchannel network in a bio-conjugated liquid-like solid (LLS) medium. Our results demonstrated successful CAR T cell migration and anti-tumor activity against CD70-expressing glioblastoma and osteosarcoma tumors. Through comprehensive analysis of cytokines and chemokines, combined with in situ imaging, we elucidated that immune recruitment occurred via chemotaxis, and the effector-to-target ratio plays an important role in overall antitumor function. Furthermore, through single-cell collection and transcriptomic profiling, we identified differential gene expression among the immune subpopulations. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach. STATEMENT OF SIGNIFICANCE: The use of specialized immune cells named CAR T cells to combat cancers has demonstrated remarkable success against blood cancers. However, this success is not replicated in solid tumors, such as brain or bone cancers, mainly due to the physical barriers of these solid tumors. Currently, preclinical technologies do not allow for reliable evaluation of tumor-immune cell interactions. To better study these specialized CAR T cells, we have developed an innovative in vitro three-dimensional model that promises to dissect the interactions between tumors and CAR T cells at the single-cell level. Our findings provide valuable insights into the complex dynamics of CAR T cell function in solid tumors, informing future research and development in this promising cancer treatment approach.

Optimization of caseinolytic and coagulating activities of Solanum tuberosum rennets for cheese making.

BACKGROUND: In recent years, the rising global demand for cheese, the high cost and limited supply of calf rennet, and consumer choices have increased research into new alternatives to animal or recombinant chymosins for cheese making. Plant proteases with caseinolytic activity (CA) and milk-clotting activity (MCA) have been proposed as alternatives for milk clotting to obtain artisanal cheeses with new organoleptic properties. They have been named vegetable rennets (vrennets). The aim of this study was to evaluate the performance of two Solanum tuberosum aspartic proteases (StAP1 and StAP3) as vrennets for cheese making and to obtain a statistical model that could predict and optimize their enzymatic activity. RESULTS: To optimize the CA and MCA activities, a response surface methodology was used. Maximum values of CA and MCA for both enzymes were found at pH 5.0 and 30-35 °C. Analysis of the degradation of casein subunits showed that it is possible to tune the specificity of both enzymes by changing the pH. At pH 6.5, the αS - and ß- subunit degradation is reduced while conserving a significant MCA. CONCLUSION: The statistical models obtained in this work showed that StAP1 and StAP3 exert CA and MCA under pH and temperature conditions compatible with those used for cheese making. The casein subunit degradation percentages obtained also allowed us to select the best conditions for the degradation of the κ-casein subunit by StAPs. These results suggest that StAP1 and StAP3 are good candidates as vrennets for artisan cheese making. © 2023 Society of Chemical Industry.

Three-Dimensional Bioconjugated Liquid-Like Solid (LLS) Enhance Characterization of Solid Tumor - Chimeric Antigen Receptor T cell interactions.

Cancer immunotherapy offers lifesaving treatments for cancers, but the lack of reliable preclinical models that could enable the mechanistic studies of tumor-immune interactions hampers the identification of new therapeutic strategies. We hypothesized 3D confined microchannels, formed by interstitial space between bio-conjugated liquid-like solids (LLS), enable CAR T dynamic locomotion within an immunosuppressive TME to carry out anti-tumor function. Murine CD70-specific CAR T cells cocultured with the CD70-expressing glioblastoma and osteosarcoma demonstrated efficient trafficking, infiltration, and killing of cancer cells. The anti-tumor activity was clearly captured via longterm in situ imaging and supported by upregulation of cytokines and chemokines including IFNg, CXCL9, CXCL10, CCL2, CCL3, and CCL4. Interestingly, target cancer cells, upon an immune attack, initiated an "immune escape" response by frantically invading the surrounding microenvironment. This phenomenon however was not observed for the wild-type tumor samples which remained intact and produced no relevant cytokine response. Single cells collection and transcriptomic profiling of CAR T cells at regions of interest revealed feasibility of identifying differential gene expression amongst the immune subpopulations. Complimentary 3D in vitro platforms are necessary to uncover cancer immune biology mechanisms, as emphasized by the significant roles of the TME and its heterogeneity.

mRNA challenge predicts brain cancer immunogenicity and response to checkpoint inhibitors.

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

Optimization of fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3) by response surface methodology.

The aim of this study was to optimize in vitro conditions to enhance fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3). The effects of StSTBc-3 concentration (0.2-5 µM), pH value (6-10) and temperature (35-50 °C) on fibrinogenolytic activity were studied through response surface methodology (RSM). We obtained a model that predicts the response accurately. The relationship between enzyme concentration and fibrinogenolytic activity was linear, while the main effect from pH and temperature on the response was quadratic. From the RSM generated model the optimum pH was 8 and the optimum temperature was 43 °C, while higher concentrations of enzyme produce higher activities. Under optimum conditions there were no statistically significant differences between the experimental responses and the ones predicted from the model. This model also predicts the activity under physiological conditions. These results confirm that StSTBc-3 is a good candidate to be considered for therapeutic uses. The generated model will be useful for biotechnological purposes.

Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana.

The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3).

Plant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and anti-platelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes ß subunit followed by partially hydrolyzed α and γ subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin ß-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.