Gasdermin D is a protein that plays a crucial role in initiating inflammation by forming pores. When it is activated, a part that inhibits its function is removed. The remaining fragments of the protein then gather together to create large pores in the cell membrane, which facilitate the discharge of inflammatory signals. Until now, studying these interactions in living cells has posed challenges, making it difficult to understand the sequence of events from oligomerization to pore creation and its integration into the membrane. A global research team, headed by the University Hospital Bonn (UKB) and the University of Bonn, has managed to shed light on this process using identified antibody fragments known as nanobodies. They are optimistic that their findings will lead to potential therapeutic uses. The research results are published in the journal Nature Communications.
Inflammasomes are complex structures in the innate immune system that activate and regulate inflammation in our bodies. A significant process initiated by these complexes is the cleavage of the protein gasdermin D (GSDMD). The active segment of GSDMD, referred to as the N-terminal domain (NTD), can create pores in the membranes of cells. This allows not only the release of pro-inflammatory cytokines but also induces pyroptosis, a type of cell death that intensifies inflammation. “However, the specifics of how and where GSDMD forms these pores, and whether this can be stopped, was previously unclear,” explains Prof. Florian I. Schmidt from the Institute of Innate Immunity at UKB, a member of the Cluster of Excellence ImmunoSensation2 and the Transdisciplinary Research Area (TRA) “Life & Health” at the University of Bonn.
To resolve these uncertainties, Prof. Schmidt’s team utilized protein inhibitors derived from specific antibodies found in alpacas. These nanobodies are significantly smaller than regular antibodies—about ten times smaller. They can attach to proteins to disrupt their functions or tag molecules for visualization. The researchers identified six different nanobodies that target GSDMD. In their experiments, they introduced the genetic instructions for two of these nanobodies into human macrophages, which are a type of white blood cell.
No pore formation in the cell membrane without oligomerization
“Our findings demonstrate that the nanobodies inhibit the formation of pores, effectively averting cell death and the release of cytokines,” states Lisa Schiffelers, the lead author and a doctoral student in Prof. Schmidt’s group at the UKB. The Bonn researchers also clarified the mechanism: the nanobodies inhibit the oligomerization of the GSDMD NTD—meaning that the individual subunits cannot come together to form larger structures. However, they do not obstruct the GSDMD NTD from inserting itself into the cell membrane. “This leads us to conclude that GSDMD NTD first embeds in the cell membrane and then oligomerizes,” adds Schiffelers. The researchers also confirmed the specific target membrane. “It turns out that GSDMD NTD integrates into the plasma membrane, which is the outermost layer of the cell, contrary to some previous assumptions that it entered the mitochondria first,” states Prof. Schmidt. Surprisingly, they found that the nanobodies also inhibit the death of macrophages when introduced externally as a purified protein. “This occurs because the initially formed pores allow nanobodies to enter the cell, after which they stop further pore formation while the cell works to eliminate existing channels,” explains Schiffelers.
The Bonn researchers, who have filed a pending patent for the GSDMD nanobodies, believe that these findings propose a potential approach where nanobodies targeting GSDMD might be utilized in treating diseases linked to pore formation and pyroptosis, such as sepsis and various autoinflammatory conditions. “Currently, our nanobodies only recognize human GSDMD and not the mouse version, thus they have yet to be tested in living organisms. Only through such tests can we truly assess their therapeutic potential,” mentions Prof. Schmidt. “In the meantime, we have also identified nanobodies against mouse GSDMD that will allow us to conduct these essential tests, and this remains a focus of our ongoing research.”
Participating institutions and funding
The study involved contributions from the UKB and the University of Bonn, as well as the Walter and Eliza Hall Institute of Medical Research (Australia) and the Whitehead Institute for Biomedical Research in Cambridge (USA). Funding for this research was provided by the German Research Foundation (DFG) via the Cluster of Excellence ImmunoSensation2 at the University of Bonn, Prof. Florian Schmidt’s Emmy Noether Research Group at UKB, and the Collaborative Research Center (SFB)1403.
Prof. Florian I. Schmidt is a cofounder of Odyssey Therapeutics, which did not take part in this study.
