How cells make their proteins – from insights to engineering

This is the final annual report from our Focus Group for Cellular Protein Biochemistry at the TUM-IAS. Matthias Feige, heading the group as a Rudolf Mößbauer Tenure Track Assistant Professor, has just obtained tenure and now continues his career as Associate Professor for Cellular Protein Biochemistry at TUM. This provides the opportunity to summarize some our achievements during the Fellowship as well to provide a few glimpses of the path ahead.

Current work in the CPB lab focuses on two major topics. The first is the quality control of membrane proteins, where principles are still mostly unknown yet have immediate biomedical relevance. Failures in membrane protein biogenesis are associated with a large number of diseases, including neurological disorders. One such example is X-linked Charcot-Marie-Tooth disease, which is caused by mutations in Connexin 32. Charcot-Marie-Tooth disease is the most common genetic disorder of the peripheral nervous system. In our work we were able to show that mutations in Connexin 32 can disturb its proper structure formation and integration into the lipid bilayer [1], a defining step for every membrane protein (Figure 1). This compromises formation of Connexin 32 gap junctions, nano-channels that allow the transport of small molecules within and between cells and are essential in our nervous system.

As a consequence, Schwann cells, which form an insulating layer around nerve cells, die and signal transmission in the nervous system is compromised. Our insights thus provide a molecular explanation for certain types of Charcot-Marie-Tooth disease, which may ultimately help in developing new therapeutic approaches. On the basis of these findings, we have meanwhile significantly expanded our scope and our team working on membrane protein biogenesis. A team of five doctoral candidates within our lab is currently performing systematic analyses on cellular quality control machinery for membrane proteins and on the structural biology of these molecular machines. This team has recently uncovered entirely new mechanisms of how the cell handles misfolded membrane proteins. These insights, some of which will be published shortly, help us in understanding at a molecular level how cells produce a major protein class that allows them to interact and build complex multicellular organisms. Also, the newly identified machines for membrane protein quality that we are analyzing may provide targets for future pharmacological treatment.

The second major research focus of the CPB lab is the biogenesis and engineering of interleukins (ILs), key sigvnaling molecules in our immune system. In humans, more than 40 different ILs exist. They constitute the language of immune cells. Depending on the individual IL, these mount, sustain, or suppress immune reactions in health and disease. Understanding interleukin biogenesis in the cell in more detail can thus provide avenues toward rationally modulating immune responses: dampening immune responses in autoimmunity, but also augmenting them when needed, e.g., to fight cancer wcells. Our work mostly focuses on the interleukin 12 (IL-12) family, which comprises four members (IL-12, IL-23, IL-27 and IL-35, Figure 2A) that span a broad range of biological functions [2] – from activating immune responses to suppressing immunity. As such, IL-12 family members are involved in a large number of human diseases including autoimmune disorders, cancer development, and sepsis [3]. Several studies from our lab in the past five years have revealed the molecular mechanisms of how cells produce IL-12 family members [4-9]. In one of these, in close collaboration with colleagues in the Bavarian NMR Center, TUM Medicine, and the Bioengineering Department at Stanford University, we were able to reveal mechanistic and structural details how cells produce IL-23 [5] (Figure 2B). This study now helps understanding how cells regulate and control protein interactions for signaling molecules (IL-23 consists of two parts), but also provides insights into this pro-inflammatory molecule that already is a successful target of antibody therapies in the clinics. In another study, in close collaboration with TUM Medicine and TUM Physics as well as with immunologists in Paris, we were able to gain detailed insights into how IL-27 acquires its native structure and how cells monitor structure formation of this protein [8]. These studies contribute to the basis of current research in the lab. Here a team of doctoral candidates and postdoctoral researchers is advancing our understanding of hitherto hardly understood IL-12 family members (namely IL-35) and is now using these insights for the rational engineering of interleukins with desired properties: Using these insights, we engineered a new human interleukin, extending the language of human immune cells. Currently we are further developing this rationally engineered biomolecule from our lab as a potential new treatment for sepsis, one of the deadliest diseases in developed countries without any good causal treatment options. Our first patent on the development of this new molecule was awarded with the Innovationspreis 2019 der BioRegionen in Deutschland. Funded by the BMBF and the Else-Kröner-Fresenius Stiftung, we have now built an interdisciplinary team in protein biochemistry, molecular immunology, pharmacology, and medicine, including an expert in the biotech business, to ultimately bring our engineered interleukins to the clinics. Within a few years, we have thus taken the step from basic science to (pre-)clinical translation, which was possible due to the broad range of expertise present at TUM.