Role of legumain in infection and inflammation

Legumain or asparaginyl endopeptidase (AEP) is a member of the CD clan of cysteine proteases and cleaves protein substrates exclusively after asparagine or (to a minor extent) aspartic acid residues. It is a highly conserved protein which is present in a large variety of animal species. It was shown that legumain has possible roles in normal lysosomal functions, antigen presentation, immune response and immune signalling, but also in apoptosis and osteoclast remodelling.

The most reliable experimental evidence for major physiological roles of legumain was obtained from animal models, where legumain null mice exhibited hemophagocytic syndrome, impaired kidney function and accumulation of macromolecules in the lysosomes which is characteristic for lysosomal storage diseases.

Recently, several reports linked legumain to various pathological conditions such as cancer and Alzheimer’s disease. However, one of the most important physiological roles of mammalian legumain is in the immune response, where it was originally thought to participate solely in the processing of foreign proteins for presentation on the MHCII complex. In the last decade it was also shown to activate TLR receptors of the innate immune system and influence signalling pathways through the processing of other membrane receptors. Contrary to many other proteases, physiological substrates of legumain were never studied on a system wide level and all known substrates were identified on a case-by-case basis. Although legumain is emerging as physiologically and clinically relevant target, due to the lack of experimental data on its substrates the majority of legumain physiological functions, especially the ones related to immune response, still remain largely unknown. This emphasizes the need to address its physiological functions by identification of its substrates, which has the potential for a major scientific breakthrough and could therefore significantly improve our understanding of legumain-dependent mechanisms behind immunity.

We recently performed a pilot proteomic analysis of macrophages from legumain null mice and our preliminary results showed that legumain ablation in macrophages caused extremely high upregulation of two peroxidases related to the antimicrobial immune response (myeloperoxidase and eosinophil peroxidase). Both peroxidases are known to produce hypochlorous acid which is a potent antimicrobial agent involved in elimination of infectous organisms. However, hypohalous acids were also reported to damage the host tissue during the inflammatory conditions such as asthma and hypereosinophilic syndrome which means that legumain could be directly involved in regulation of those processes, but the molecular mechanisms remain unknown.

The data obtained in our preliminary experiments demonstrated that the proposed research can provide an explanation of legumain-null phenotype at the molecular level. Moreover, this project enables a novel and unique insight into the role of legumain in immune response and inflammation, as well as on the regulation of immune response and inflammation in general. In this proposal, we plan to expand our research by performing a systematic proteomic analysis of several tissue types of young and aged mice in order to obtain a detailed understanding of how legumain regulates physiological processes at the molecular level. The obtained results will be supplemented with quantitative PCR and antibody array data and will be used for biochemical and biological validations of the identified molecular processes and pathways. Finally, the in vivo role of legumain in defense against pathogen infection will be examined in animal models. Legumain dependant effect on antimicrobial action of immune cells through the generation of hypochlorous acid could open new venues for development of therapeutic approaches to fight infection.

ARRS project: J7-9435, Marko Fonović

Duration: 1. 7. 2018 – 30. 6. 2021

Role of cysteine cathepsins in complement activation in cancer

There is still a significant unmet need for successful early diagnosis and treatment of major life-threatening or debilitating diseases like cancer and other inflammation-associated diseases, making this project of high biomedical relevance. One of the major reasons is that these diseases are still not well understood at the molecular level. The same is true also for the role of cysteine cathepsins and complement in these diseases. Using a combination of biochemical, proteomic and in vivo experiments, important new information will be obtained that will shed light on cancer progression and on the role of cysteine cathepsins in these diseases, including in complement regulation. Moreover, the project is expected to open new avenues in the area of of complement research, which recently began to emerge as one of the key factors in development of tumorigenesis. Identification of C1q ligands on the surface of cancer cells would be the real highlight of the project and a major breakthrough in all these areas of research, including complement, cancer and proteolysis. The gained knowledge will thus not only significantly contribute to our understanding of the complex biological phenomena, but will also be instrumental in biomedical research to understand and develop novel strategies to combat cancer and other inflammation-associated diseases.

ARRS project no. J1-1710, Boris Turk

Duration: 1. 7. 2019 – 30. 6. 2022