Dr. Livija Tusar
Researcher at Jozef Stefan Institute
+1
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Academic Career Length
6Years (2021-Present)
(Calculated once Scilit publication data is synced: current year − year of first publication + 1)
Representative Publications
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Journal Article
Livija TusarAUAleksandra Usenik
Aleksandra UsenikBTBoris Turk
Boris TurkDTDusan Turk
Dusan TurkProtein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.
24 April 2023 in Communications Biology
Journal Article
Livija TusarJLJure Loboda
Jure LobodaFIFrancis Impens
Francis Impens
PSPiotr Sosnowski
Piotr SosnowskiEQEmmy Van Quickelberghe
Emmy Van Quickelberghe
DTDusan Turk
Dusan Turk +11
Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.
Journal Article
For risk communication, it is important to understand the difference between “hazard” and “risk”. Definitions can be found in Codex Alimentarius and the European Union (EU) General Food Regulation (EC) No. 178/2002. The use of these terms as synonyms or their interchange is a recurrent issue in the area of food safety, despite awareness-raising messages sent by EFSA (European Food Safety Authority) and other interested entities. A quick screening of the EU’s food regulations revealed several inconsistencies. Hence, it was considered necessary to further investigate if regulations could act as a source for this problem. A software tool was developed to support the detection and listing of inconsistent translations of “hazard” and “risk” in certain EU food regulations. Subsequently, native-speaking experts working in food safety from each EU country were asked to provide their individual scientific opinion on the prepared list. All data were statistically analysed after applying numerical scores (1–5) describing different levels of consistency. Results showed that the most common problem was the interchange of “hazard” with “risk” and vice versa. This lack of consistency can create confusion that can further translate into misjudgments at food risk assessment and communication levels.
Preprint
For risk communication, it is important to understand the difference between "hazard" and "risk". Definitions can be found in Codex Alimentarius and the European Union (EU) General Food Regulation (EC) No 178/2002. The use of these terms as synonyms or their interchange is a recurrent issue in the area of food safety, despite awareness-raising messages sent by EFSA (European Food Safety Authority) and other interested entities. A quick screening of EU’s food regulations revealed several inconsistencies. Hence, it was considered necessary to further investigate if regulations could act as a source for this problem. A software tool was developed to support the detection and listing of inconsistent translations of “hazard” and “risk” in certain EU food regulations. Subsequently, native-speaking experts working in food safety, from each EU country, were asked to provide their individual scientific opinion on the prepared list. All data was statistically analyzed after applying numerical scores (1-5) describing different levels of consistency. Results showed that the most common problem was the interchange of “hazard” with “risk” and vice versa. This lack of consistency can create confusion that can further translate into misjudgments at food risk assessment and communication level.
26 October 2026 in Biological Chemistry
Journal Article
Livija TusarDJDavid Jurnecka
David JurneckaTUTadej Uršič
Tadej Uršič
DTDusan Turk
Dusan TurkCysteine cathepsins are receiving large attention due to their widespread participation in physiological and pathological processes. The link between their specificity and biological function is more complex than previously anticipated. Structural analysis revealed that this complex specificity is anchored in the architecture of the active site cleft. Unlike classical proteases with well-defined specificity pockets S1, the corresponding P1 residue of a cathepsin substrate points toward solvent, precluding a clear P1-driven specificity profile. This review analyzes the evolution of cysteine cathepsin research, spanning from the initial biochemical era and the “golden age” of structural biology to contemporary high-throughput profiling, degradomics, and computational analysis. The surge in available data has reshaped our understanding, shifting from rigid active-site models toward statistically driven patterns that reflect the inherent flexibility of structure. For cysteine cathepsins, the next frontier lies in discerning redundant versus unique physiological and pathological roles. This requires integrating high-resolution localization profiles, adequate prediction models, and in situ measurements of degradation products. Such integration will mark a new era of proteolysis studies, replacing individual enzyme-centric research with a multidimensional synthesis of diverse data sources encompassing the expanding landscape of proteases and their substrates.
Journal Article
MZMiki Zarić
Miki ZarićLivija TusarLKLovro Kramer
Lovro Kramer
Olga VasiljevaMNMatej Novak
Matej NovakBTBoris Turk
Boris Turk +4
Cathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (KD = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions.
About Me
Livija Tusar (birth name Lah) received master's degree in 1990 and PhD in 1998 from Faculty of chemistry and chemical technology, University of Ljubljana, Slovenia. In period 1987 to 1996, she was researcher at National Institute of Chemistry in Ljubljana involved in postgraduate research program for young researchers funded by Slovenian Research Agency. In 1990, she received Slovenian national award “Boris Kidric fund for inventions” with co-workers Bohanec S., Tusar M., Meglic M., Ljubic T. for “C13 SYS - CARBON computer software for expert solving of 13C NMR spectra problems”. Then she was independent researcher until 1999, Counsellor to the Slovenian government until 2004, and Undersecretary at the Ministry of higher education, science and technology until 2006. In period 2006 to 2010, she was researcher in Department for Pharmacokinetic Studies, Development Center Slovenia, LEK Pharmaceuticals d.d., a Sandoz company.
Since 2006, she has been assistant professor for food safety lecturing Statistical design of experiments and modelling at the University of Maribor, Faculty of agriculture and life sciences. Since 2010, she has been part time General director in Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins and part time a researcher in the group for Structural biology, led by Prof Dr Dusan Turk, in Department of biochemistry and molecular and structural biology at Jozef Stefan Institute, Ljubljana, Slovenia.