Supplementary data for the article: Raskovic, B.; Lazic, J.; Polovic, N. Characterisation of General Proteolytic, Milk Clotting and Antifungal Activity of Ficus Carica Latex during Fruit Ripening. Journal of the Science of Food and Agriculture 2016, 96 (2), 576–582. https://doi.org/10.1002/jsfa.7126

Supplementary material for: [https://doi.org/10.1002/jsfa.7126]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2017]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3397

Ubikvitinom posredovana degradacija unutarćelijskih proteina

Eukaryotic cells, from yeast to human, contain some 6000 to 30000 protein-encoding genes and at least as many proteins. While much attention and research had been devoted to how proteins are synthesized, the reverse process, i.e. how proteins are degraded, long received little attention. The Nobel Prize in Chemistry for 2004 is shared between three scientists: Aaron Ciechanover, Avram Hershko and Irwin Rose who discovered ubiquitin-mediated proteolysis. Numerous cellular processes regulated by ubiquitin-mediated proteolysis include the immune response, cell cycle, DNA repair and transcription and protein quality control

Ubikvitinom posredovana degradacija unutarćelijskih proteina

Eukaryotic cells, from yeast to human, contain some 6000 to 30000 protein-encoding genes and at least as many proteins. While much attention and research had been devoted to how proteins are synthesized, the reverse process, i.e. how proteins are degraded, long received little attention. The Nobel Prize in Chemistry for 2004 is shared between three scientists: Aaron Ciechanover, Avram Hershko and Irwin Rose who discovered ubiquitin-mediated proteolysis. Numerous cellular processes regulated by ubiquitin-mediated proteolysis include the immune response, cell cycle, DNA repair and transcription and protein quality control

Supplementary data for the article: Milošević, J.; Janković, B.; Prodanović, R.; Polović, N. Comparative Stability of Ficin and Papain in Acidic Conditions and the Presence of Ethanol. Amino Acids 2019, 51 (5), 829–838. https://doi.org/10.1007/s00726-019-02724-3

Supplementary material for: [https://link.springer.com/article/10.1007/s00726-019-02724-3]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3932

Supplementary data for the article: Milošević, J.; Janković, B.; Prodanović, R.; Polović, N. Comparative Stability of Ficin and Papain in Acidic Conditions and the Presence of Ethanol. Amino Acids 2019, 51 (5), 829–838. https://doi.org/10.1007/s00726-019-02724-3

Supplementary material for: [https://link.springer.com/article/10.1007/s00726-019-02724-3]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3932

Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development

The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group’s reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases

Supplementary data for the article: Vatić, S.; Mirković, N.; Milošević, J. R.; Jovčić, B.; Polović, N. Đ. Broad Range of Substrate Specificities in Papain and Fig Latex Enzymes Preparations Improve Enumeration of Listeria Monocytogenes. International Journal of Food Microbiology 2020, 334, 108851. https://doi.org/10.1016/j.ijfoodmicro.2020.108851

Supplementary material for: [https://doi.org/10.1016/j.ijfoodmicro.2020.108851]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4090

Supplementary data for the article: Milošević, J.; Petrić, J.; Jovčić, B.; Janković, B.; Polović, N. Exploring the Potential of Infrared Spectroscopy in Qualitative and Quantitative Monitoring of Ovalbumin Amyloid Fibrillation. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2020, 229. https://doi.org/10.1016/j.saa.2019.117882

Supplementary material for: [https://doi.org/10.1016/j.saa.2019.117882]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3884]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3895

One-step purification and freeze stability of papain at acidic pH values

Papain is a proteolytic enzyme of great commercial value. It is a cysteine protease highly expressed in Carica papaya fruit latex, but also present in papaya leaves. Purification procedures mostly deal with the latex and include a combination of precipitation and/or chromatographic techniques. Due to its solubility, structure and activity characteristics, the pH and salt content play significant roles in handling papain extracts. Here we report a simple, rapid and easily scalable procedure for papain purification from papaya leaves, which contain different contaminants as compared to papaya latex. Sodium chloride precipitation of contaminants at pH 5 followed by ammonium sulphate precipitation resulted in the removal of other leaf proteins and protein fragments from papain solution and about a 3-fold purification. The procedure also benefits from the suppression of autoproteolysis and preservation of the native structure, as confirmed by FTIR analysis, and the high recovery of activity of over 80%

On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4391