Proteolysis and its regulation in health and disease

Proteolytic processing is one of the most important irreversible posttranslational protein modifications, since it affects a great variety of physiological processes critical for life, including the immune response, cell cycle, cell death, and protein and organelle recycling. Proteases are strictly controlled and imbalances in their activities have been found to be critical in a number of pathologies, such as inflammation, cancer, infectous and neurodegenerative diseases, thereby suggesting proteases as valuable drug targets. Especially in inflammation, which plays a major role in onset and progression of many diseases proteases, in particular cysteine cathepsins, caspases, and metalloproteases have very important roles. As in most of these diseases there is an urgent clinical need for improved diagnosis, therapies and patient welfare, the major focus of our program will be on cysteine cathepsins, for which unambiguous evidence for their major roles in these diseases has been provided using genetic ablation and pharmacological inhibition in animal models. In addition, caspases, legumain, various metalloproteases, and aspartic cathepsins will be investigated. However, understanding the precise role of an individual protease in a disease remains a major challenge for successful therapeutic applications. The major goals of our program are therefore: (i) to further understand molecular signaling pathways by which selected proteases regulate physiological processes in health and disease and how they are regulated, and (ii) use this knowledge for possible therapeutic interventions in disease, in particular minimally invasive diagnostic imaging and targeted drug delivery, with some work devoted to small molecule inhibitor development. To understand the signaling pathways, we would further focus on identification of physiological substrates of investigated proteases and their validation in vitro and in vivo, as well as biochemical and structural characterization of their interaction. A breakthrough can be achieved by novel multimodal imaging systems for diagnostic imaging based on Sybodies and novel targeted drug delivery systems with theranostic potential. Moreover, the identified protease substrates in the disease context have a biomarker potential, which will be explored in clinical samples. We therefore believe that this research will enhance our understanding of protease signaling pathways and their regulation in health and disease progression at the molecular level, our ability to diagnose disease and monitor its progression, and improve the existing therapeutic opportunities, which would take personalized medicine to the next level. We feel this is extremely important in the current situation, where because of the COVID-19 pandemic, a number of diseases, especially cancer were not given proper attention and many patients with advanced disease are expected to be diagnosed, requiring best possible therapies for favourable outcome.

Proteolytic enzymes play a key role in intra- and extracellular protein processing and degradation, which are among the most important physiological processes. Under normal conditions proteolysis is a highly regulated process. However, when regulation fails, proteases can be extremely harmfull for the organism, leading to numerous diseases (Turk 2006; Drag and Salvesen, 2010; Turk et al., 2012). In the past, our research group has contributed significantly to the development of the field with the discoveries of several cathepsins and their endogenous inhibitors, lysosomal pathway to apoptosis as well as pioneering efforts in the fields of development of novel nanoparticle-based targeted drug delivery systems, allosteric protease inhibitors and novel activity-based probes for proteases which classify our research at the cutting edge in the field. In the last financing period, we were the first to demonstrate that cathepsins can act as sheddases in cancer and identified a number of novel protease substrates, thereby enabling identification of their physiological roles at the molecular level. This was tihghtly linked with the development of novel proteomic approaches in the lab. In addition, we promoted the idea of cathepsins as suitable targets for targeted drug delivery and were the first to show that we can use endogenous inhibitors and antibody-like molecules for their targeting. Finally, we contributed significantly to the development of novel minimally invasive diagnostic imaging approaches based on small molecule imaging of proteases. This is reflected also in high citation of our works in this competitive field. The proposed research program offers new possibilities for further understanding of protease regulation, including its role in inflammation-associated diseases (cancer, neurodegeneration,infectious diseases including COVID-19, etc.), as well as possibilities for rational drug design, biomarker discovery and other novel diagostic and therapeutic opportunities, including development of novel theranostic systems on a way towards personalized medicine. Therefore, we have good arguments to expect that the proposed research will be accomplished succesfully also in the next funding period. A major interest of the pharmaceutical companies for proteases including the cathepsins is supporting the idea that this problematic is extremely important. It can be concluded that the proposed research topics belong among the most attractive areas of research in the fields of biomedicine, agriculture and pharmacy, to list just some of them. A further confirmation for the high level of scientific achievements of the group is evident from numerous publications in the most important international journals, including 28 in top 5% journals and consequently the high citation of the works of the PI (over 20500 citations total, H-index 60) and the group. Moreover, this is evident also from the numerous awards received by research group members as well as from the list of international collaborations with a number of top class researchers worldwide.

Although the proposed research is basic research, it also has its applied component and can be classified as strategic basic research. Members of the group have extensively collaborated with Slovene (Lek, Krka, Acies Bio, …) and foreign industry, which resulted in a significant amount of contract-based research, as well as extensive collaborations within the Center of Excellence CIPKEBIP, which was financed by European Structural Funds. Moreover, as a result of the work several international patents have been filed and/or granted, all in the field of targeted drug delivery and minimally invasive diagnostic imaging, thereby offering new opportunities for translational medicine. The work also offers great opportunity for students to be trained in the most advanced methods and areas, such as proteomics (the only proteomics facility in Slovenia), chemogenomics, targeted drug delivery and in vivo imaging (the only in vivo imaging system in Slovenia). All these fields have namely high international priority as they are of extreme importance in modern drug discovery and early diagnostics, and combining them should help translate the findings into clinics and bring the personal medicine to a new level. It is therefore not suprising that research within this programme since its beginning lead to over 300 finished BSc/MSc theses (including 67 stage 2. bologna MSc), and over 70 PhD theses, including 21 in the last programme call since 2015. After finishing their studies, numerous researchers left the group and went to other institutes and universities. At the University of Ljubljana (UL) researchers from this programme form the core of the biochemistry program and chair of biochemistry at the Faculty of Chemistry and Chemical Engineering (FKKT). A number of the researchers from the programme went to pharmaceutical industry (over 10 in last 7-8 years). Investigations like this combining biochemistry, chemogenomics, proteomics, structural biology and molecular and cell biology are critically important for the development of modern biotechnology and biomedical research. Excellent and up-to-date science and its transfer to modern technology are of major importance for the sustainable socio-economic development and competitiveness of Slovenia and its classification among the developed members of EC. In addition, members of the project team have received wide-spread international recognition, which is very important for the world-wide promotion of Slovenia and as such also for preservation of national identity of Slovenia. The group organized a number of international meetings with participation of numerous excellent foreign researchers from academia and industry, including several Nobel laureates. A number of international collaborations have been established, which all contribute to the international reputation of Slovenia. Dr. Boris Turk is currently a member of EMBO Fellowship Committee and FEBS Publishing Committee, and was vice President of EMBC (2017-2020), Secretary General of European Cell Death Organization (ECDO) (2008-2018), he was councilor of the International Proteolysis Society (IPS, 2001-2005; 2009-2013) andt its Secretary (2009-2011) and President (2011-2013), he is an elected member of EMBO and Academia Europea (London) and was awarded Zois award for outstanding achievements in the field of Protease signaling in 2011. Dr. Vito Turk, now retired but still active group member, was performing leading functions in the Federation of European Biochemical Societies (FEBS) and in IUBMB, he is a member of Slovene Academy of Sciences and Arts, EMBO, European Academy (London), etc. In addition, other group members also received various awards and amendments. Furthermore, several group members serve as Editors or Editorial board members of various international journals, which additionally contributes to international recognition of Slovenia. With our achievements we contribute also to the cultural rise of Slovenia as both science and arts are indispensable for that.