Atypical antipsychotic clozapine binds fibrinogen and affects fibrin formation

Clozapine is an atypical antipsychotic used for the treatment of schizophrenia. The prescribed target daily doses may reach 900 mg. Literature studies report a connection between clozapine usage and thrombosis development. Our in vitro study aimed to provide insight into molecular bases of this observation, investigating clozapine binding to fibrinogen, the main plasma protein involved in hemostasis. Fibrinogen/clozapine interaction was confirmed by protein fluorescence quenching, with an affinity constant of 1.7 × 105 M−1. Direct interactions did not affect the structure of fibrinogen, nor fibrinogen melting temperature. Clozapine binding affected fibrin formation by reducing coagulation speed and thickness of fibrin fibers suggesting that in the presence of clozapine, fibrinogen may acquire thrombogenic characteristics. Although no difference in fibrin gel porosity was detected, other factors present in the blood may act synergistically with altered fibrin formation to modify fibrin clot, thus increasing the risk for development of thrombosis in patients on clozapine treatment. ORAC and HORAC assays showed that clozapine reduced free radical-induced oxidation of fibrinogen. All observed effects of clozapine on fibrinogen are dose-dependent, with the effect on fibrin formation being more pronounced.This is the peer-reviewed version of the article: Gligorijević, N.; Vasović, T.; Lević, S. M.; Miljević, Č.; Nedić, O.; Nikolić, M. Atypical Antipsychotic Clozapine Binds Fibrinogen and Affects Fibrin Formation. International Journal of Biological Macromolecules 2020, 154, 142–149. [https://doi.org/10.1016/j.ijbiomac.2020.03.119

Antipsychotic clozapine binds catalase and preserves its activity in oxidative environment

Oxidative stress undoubtedly accompanies mental disorders, and the pleiotropic effects of atypical antipsychotics, recommended drugs in the treatment of psychosis, are not clarified at the molecular level. Catalase is one of the key enzymes of the primary antioxidant protection system. This work studied the binding of second-generation antipsychotic drug Clozapine to commercial bovine liver catalase. Using various spectroscopic methods under simulated physiological conditions, we found moderate binding affinity of clozapine for catalase (Ka ~ 2x105 M-1), the binding influenced the secondary and tertiary structure of protein (according to UV-VIS and CD spectroscopy) and it managed to slightly increase its thermal stability. In AAPH induced oxidation experiments, we found that clozapine efficiently protects catalase from free-radicals oxidation and preserves its activity. Clozapine affects catalase activity in dose dependant manner, having no significant effect at lower concentrations but significantly inhibiting enzyme at saturating concentrations. In conclusion, our results indicate that the effect of direct binding of clozapine to catalase can be both beneficial and harmful and that this effect is dose dependent.The Biochemistry Global Summit, 25th IUBMB Congress, 46th FEBS Congress, 15th PABMB Congress, July 9-14, 2022, Lisbon, Portuga

Expression of SARS-CoV-2 spike protein receptor binding domain in mammalian cell culture

Since the occurrence of SARS-CoV-2 in the late 2019/early 2020, there has been a huge demand for SARS-CoV-2 related reagents, including high quality viral antigens. The immunostimulating SARS-CoV-2 antigens, spike (S) and nucleocapsid (N) protein, were of particular importance for the production of the serological assays which measure the level of the natural infection or vaccine generated viral specific antibodies in human samples. Among other S protein variants, our team focused on the production of its receptor binding domain (RBD) for the purpose of detecting potentially neutralizing antibodies which could prevent (re)infection by reducing viral entry into ACE-2 expressing host cells. His tagged RBD (S protein amino acid range of 319-541) was expressed in HEK293T cells, using polyethylenimine reagent as the plasmid carrier. Protein expression was monitored by SDS-PAGE and Western blot using anti-His antibodies. Protein purification was achieved in one step by immobilized metal affinity chromatography (IMAC). RBD sequence was identified by mass spectrometry with the sequence coverage of over 80%, including 14 unique peptides. Extensive protein glycosylation was noticed, RBD travelled as a 37 kDa protein in SDS-PAGE. Finally, RBD immunoreactivity was proven in ELISA assays with Covid negative and Covid positive serum samples. Here we report an inexpensive and efficient method for the production of small scale quantities of high quality RBD of SARS-CoV-2 S protein, which could be used in immunological and biochemical assay. Scaling-up is possible with the right choice of culturing conditions.Konferencijski prilog podržan UNDP projektom Tanje Ćirković Veličković, a publikacija u okviru aktivnosti na projektu FoodTwin Symposium-a

Production of the protein, probable marker for linden allergy

Pollen allergies have tremendous global clinical impact. Diagnosis of allergy in a clinical setting is based on the use of purified allergens which are considered to be more accurate and sensitive than crude pollen extracts. Thus, proper identification and characterization of allergens at the molecular level and production of a recombinant counterpart are of outmost importance for the efficient use of the current diagnostic tools for allergy. Recombinant allergens are nowadays produced instead of natural, as isolation of a natural allergen is a troublesome process. Tree pollens that are most commonly associated with respiratory allergies are produced by birch in the Northern, Central, and Eastern Europe, and by olive and cypress in the Mediterranean regions. Accordingly, most research so far has focused on the allergenicity of pollens from birch, olive and cypress. Allergy to linden pollen has been known, but no allergen has been described from this source yet. Here we propose cloning and production of the first recombinant linden pollen allergen that can be used as a marker allergen of linden pollen allergenicity and complement existing diagnostic allergen-arrays. Also, production of this recombinant allergen has potential to contribute to the development of novel allergen-diagnostic tools through collaborations with biotech companies

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties

Related to research data no. 1: [https://cherry.chem.bg.ac.rs/handle/123456789/6465]Related to research data no. 2: [https://cherry.chem.bg.ac.rs/handle/123456789/6469

Biocorona formation of hen egg white proteins onto the surface of polystyrene and polyethylene terephthalate

Ovalbumin (OVA), a main protein of egg white, has characteristic structural fold of a serpin-family of proteins, propensity to fibril formation and stability to digestion. Microplastics (MPs) contaminating our food can interact with food proteins in the food matrix and during digestion. In this study adsorption of OVA to polystyrene (PS) (110 μm and 260 μm), polyethylene terephthalate (PET) (140 μm) MPs were investigated in acidic (pH 3) and neutral (pH 7) conditions. Formations of corona on MPs were investigated using isolated OVA and egg white protein extract comparatively. OVA adsorption depends on MPs size, polymer chemistry and pH, being highest in acidic pH and higher for PS. Adsorption of OVA to PS and PET reaches dynamic equilibrium after 4h resulting in disruption of tertiary structure and formation of hard and soft corona around MPs. Shorter fragments of OVA populate hard corona, while soft corona exclusively consist of full length OVA, albeit in its non-native conformation. The conformational changes resemble those induced by heat treatment with re-arrangement of α-β secondary structures. Structural changes are striking for the OVA in corona around MPs. Soft corona OVA preserves thermal and proteolytic stability, but loses ability to form fibrils upon heating. OVA is abundantly present in corona around MPs also in the presence of other egg white proteins. MPs contaminating food may bind and change structure and functional properties of main egg white protein

Identification of isoforms of shelfish tropomyosin

rom over 10 kg in 1960 to over 20 kg in 2014, the yearly per capita consumption of marine products has increased significantly during the past 50 years. In many nations, especially those with poor overall protein intake, seafood protein is a crucial component of the diet1. However, as defined by the European Community shellfish protein tropomyosin (TPM) is one of the major allergens and major causes of anaphylaxis 2. Although TPM originating from vertebrates is not considered as an allergen there is evidence that several fish tropomyosin can be allergenic3. TPM protein is organized of two parallel alpha-helical molecules which are wound around each other forming a coiled -coil structure2. Although the degree of similarity between TPM molecules is high, their allergenic potency is different. Scientists putting a lot of effort into iden tifying and sequencing tropomyosin isoforms since this information probably explains the intriguing nature of the TPM molecule. This is a very challenging task given that the differences in mass and pI values between TPM isoforms are discrete. TPM was isolated from mussels (Mytilus galloprovincialis), and clams (Venerupis philippinarum) according to the protocol developed within/and for purposes of the IMPTOX research project. The obtained “in-house” TPM proteins were resolved using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Isoelectric focusing was performed on rehydrated Immobiline strips IPG 3-5.6NL 13 cm in Ettan IPGPhor 3 device. The second dimension was carried out on 12% PAA gels. Upon protein bands excision to prevent TPM interchain disulfide cross-linking reduction and alkylation of cysteine was performed. The samples were digested with proteomics-grade trypsin in a 1:50 enzyme-to- substrate ratio overnight at 37 °C. The obtained peptides were chromatographically separated using the EASY-nLC II system and analyzed using Orbitrap Exploris 240 mass spectrometer. 2D-PAGE resolved two isoforms of mussel TPM and up to eight isoforms of clam TPM. The determined pI value of the dominant isoform of mussel TPM was 4.7 while the discrete band arising from the second TPM isoform was slightly shifted toward acidic pI and smaller molecular mass. The determined pI value of the three most dominant clam TPM isoforms was 4.8, the fourth isoform was slightly shifted toward a more basic pI value and lower protein molecular weight. The rest of the isoforms were slightly shifted toward more acidic pI and at a similar molecular weight as the three dominant isoforms

Supplementary data for the article: Vujatović, T. B.; Vitorović-Todorović, M. D.; Cvijetić, I.; Vasović, T.; Nikolić, M. R.; Novaković, I.; Bjelogrlić, S. Novel Derivatives of Aroylacrylic Acid Phenylamides as Inducers of Apoptosis through the ROS-Mediated Pathway in Several Cancer Cell Lines. Journal of Molecular Structure 2022, 1250, 131702. https://doi.org/10.1016/j.molstruc.2021.131702.

Supplementary material for: [https://doi.org/10.1016/j.molstruc.2021.131702]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4761

Atypical antipsychotic clozapine binds fibrinogen and affects fibrin formation

Clozapine is an atypical antipsychotic used for the treatment of schizophrenia. Prescribed daily doses of clozapine may reach over 900 mg/day. Some studies reported a connection between clozapine usage and the development of thrombosis. Our in vitro study aimed to provide insight into molecular bases of this observation, investigating clozapine binding to isolated fibrinogen, the main protein involved in hemostasis. Fibrinogen/clozapine interaction was confirmed by protein fluorescence quenching, with affinity constant calculated to be 1.7 9 105 M1 and the number of binding sites more than one. Direct interactions do not affect the structure of fibrinogen, as determined by UV-VIS spectrometry and Fourier-transform infrared spectroscopy, nor fibrinogen melting temperature, examined by fluorescence spectroscopy. However, clozapine binding affected fibrin formation, by reducing coagulation speed and thickness of fibrin fibers. This behavior suggests that in the presence of clozapine, fibrinogen may acquire thrombogenic characteristics. Although no difference in fibrin gel porosity was detected, other factors present in the blood may act synergistically with altered fibrin formation to modify fibrin clot, thus increasing the risk for development of thrombosis in individuals on clozapine treatment. By ORAC and HORAC antioxidant assays, we found that clozapine efficiently protects fibrinogen from free-radicals oxidation. Since the effect of clozapine on fibrin formation is dose-dependent, it seems that the dosage of the medication could be the main factor that determines if clozapine will have a more positive or negative effect on fibrinogen and coagulation process in vivo

Trypsin as a proteomic probe to assess food protein digestibility in relation to post- translational modifications

Background: We have undertaken study on our porcine-derived trypsin generated proteomic data of the major peanut allergen Ara h 1 from the raw and roasted peanut, to assess possible facilitating/hindrance effects on trypsin digestion efficacy caused by post- translational and chemical modifications (PTMs) positioned on K/R residues. If potential hindrance effects caused by PTMs are observed with porcine trypsin, then they can be just augmented and more pronounced within human intestinal digestion. The logic for such reasoning is in inferior performance of human trypsin compared to porcine-derived used in proteomic digestion protocols, also the lower trypsin-to-sample ratio and much shorter digestion times, even though gastric digestion precedes and trypsin is not the sole digestive enzyme. Methods: Novel method was developed to decipher outcomes at scissile bonds using PEAKS Studio-X+ in reassessment of high-resolution tandem mass spectrometry data on 18h-long trypsin digestion protocol. Results: In eight modified K/R residues involving methylation, dihydroxy and formylation, differences in extent of miscleavage between modified and unmodified peptides, were significantly higher (>10%) in modified peptides. Conclusion: It is important to elucidate impact of modifications on trypsin digestion performance, but also on other proteases involved in digestion process due to possible effects on allergenicity of food proteins/peptides.Related to Book of Abstracts: [https://www.cost-infogest.eu/content/download/4051/35805/file/V-ICFD%20Book%20of%20Abstracts.pdf]Related to record of lecture: [https://www.youtube.com/watch?v=Dj0_1fyY724