Functional principles of legumain inhibition by macrocypin

Enzymes are important signaling molecules that are involved in virtually all biological processes in all living organisms. Within the huge repertoire of enzymes, proteases represent an important sub-family, which fulfills their signaling function by specifically cleaving other proteins. Signal transduction must be regulated tightly and on different levels to ensure that the right reaction takes place at the right time. Wrong signals can result in severe disorders like cancer or neurodegenerative diseases. One example of a protease that is especially important for our immune system is legumain. Legumain cleaves foreign proteins in a way so that they can be recognized by our immune system. Consequently, an immune reaction is initiated to protect ourselves from foreign dangers (e.g. bacteria). However, too high levels of, or mislocated, legumain have been linked with disorders like breast cancer and Alzheimers disease. Clearly, legumain activity must be regulated tightly to prevent pathologic disorders. Nature established different strategies to control its function. One very efficient way is by the development of specific proteins that bind to legumain and thereby prevent it from cleaving other proteins. These regulatory molecules are termed inhibitors. Only two classes of natural legumain inhibitors have been identified yet. One of which are macrocypins from the parasol mushroom. This class of proteins is especially interesting to us, since they are known as exceptionally stable proteins tolerating extremes of pH and temperature. Because of their special properties they represent an attractive target for biotechnological applications like the design of bioactive molecules. Within the project Functional principles of legumain inhibition by macrocypin we aim to study how macrocypins interact with legumain on a molecular and mechanistic level. To that end, we will combine biochemical, biophysical and structural techniques to get an in-depth understanding of their interplay with legumain. The results of the here described proposal will allow to develop legumain- reactive molecules based on a completely new template and with further application in medical and biotechnological fields.

Enzymes are important molecules that play crucial roles in all living organisms. Proteases are a specific type of enzyme that acts as signaling molecules by cutting other proteins. Signal transduction must be regulated tightly and on different levels to ensure that the right reaction takes place at the right time. Wrong signals can result in severe disorders like cancer or neurodegenerative diseases. One example of a protease that is especially important for our immune system is legumain. Legumain cleaves foreign proteins in a way so that they can be recognized by our immune system. Consequently, an immune reaction is initiated to protect ourselves from foreign dangers (e.g. bacteria). However, too high levels of, or mislocated, legumain have been linked with disorders like breast cancer and Alzheimer's disease. Clearly, legumain activity must be regulated tightly to prevent pathologic disorders. Nature has developed different ways to control legumain's function, and one effective method is through specific proteins called "inhibitors". These inhibitors bind to legumain and prevent it from cutting other proteins. One interesting group of inhibitors comes from the parasol mushroom and is known as "macrocypins". They are stable even under extreme conditions which makes them attractive starting points for biotechnological applications. The research project "Functional principles of legumain inhibition by macrocypin" focused on understanding how macrocypins interact with legumain at the molecular level. Within this project, we successfully determined the three-dimensional structure of legumain when bound to various macrocypins, providing a detailed view of their interaction. The findings unveiled the precise mechanism by which macrocypins bind to legumain, transforming them from substrates (proteins that are typically cleaved by enzymes) into effective inhibitors. Additionally, we found species-specific differences in binding among various macrocypins, which will have implications for their potential use as therapeutic agents. Notably, some macrocypins form covalent interactions with legumain, a strong and stable type of bond that further enhances their inhibitory activity. Furthermore, we found that certain isoforms of macrocypins inhibit other cysteine proteases, indicating that they might serve as previously unknown broad-spectrum inhibitors. In summary, our studies uncovered the potential of macrocypins as a new scaffold for drug development, providing the basis for the design of specific legumain inhibitors.