New detection method for protein degraders

The research team from MPI of Biochemistry and the University of Waterloo have developed a new way to detect those CRLs in the fleet that are "on”, which reveals the CRLs deployed to solve cellular stresses and to perform the actions of some anti-cancer drugs. First author Lukas Henneberg explains: "If a cell is exposed to an increase in iron or to infectious bacteria, the proteins that would further increase iron to toxic levels or that would prevent the immune response from curing the infection must be destroyed. Such proteins are targeted by the CRL destroyers" by tagging them with ubiquitin by Cullin-RING Ligases.

The fleet of CRLs essentially sail around in the inside of the cell, with each one individually waiting for a signal that it is needed. When the signal occurs, the needed CRL is temporarily switched on by attachment of another protein, called NEDD8. As soon as the destructive action of the CRL is no longer needed, the CRL is turned off by removal of NEDD8. The researchers from the laboratories of Brenda Schulman, Max Planck Institute (MPI) of Biochemistry, and Sachdev Sidhu, University of Waterloo, have now presented the first of a two-step method to detect which CRLs are attached to NEDD8, and thus are switched on.

They produced a synthetic antibody recognising CRL-molecular-machines attached to NEDD8. The researchers determined a crystal structure, essentially a 3D molecular photograph, showing how the antibody can capture NEDD8 attached to almost all CRLs, only when a CRL is switched on to allow ubiquitin tagging of proteins to be destroyed. Thus, the synthetic antibody is an activity-based probe, or a “molecular radar”, that can detect which CRLs are activated to tag their target proteins for destruction.

The scientists then developed the second step of the new method to find out which of the entire fleet of CRLs are switched on under normal cellular conditions, and which are switched on to adapt to changing cellular needs. The CRL molecular machines bound to the antibody, i.e. the active ones, were removed from the cells and collected in order to use state-of-the-art mass spectrometry to measure which and how many CRLs were active in the cells at a specific point in time.
In the current study, the authors identified, which CRLs are turned on in response to iron, and which are turned on by cellular signs of inflammation. The authors also studied CRLs that are turned on for the action of so-called "degrader" drugs. Degrader drugs are therapies that cause a CRL to target a disease-causing protein for destruction. For now, degrader drugs are used to treat some cancers, although the concept is being investigated for other diseases. The new method showed that the available amount of certain CRLs varies in different cell types, which influences the effectiveness of the degrader drugs. The more CRL "destroyers" are already switched on in a cell, the faster a degrader molecule can cause the disease causing protein to be eliminated.

The researchers also teamed up with Peter Murray’s lab at the MPI of Biochemistry to study the active CRLs in macrophages. Comparing the active CRL molecules of macrophages specialised for fighting bacteria with those of macrophages specialised for wound healing revealed clear differences that suggest the types of adaptations required for macrophage cells to do these very different functions.

The findings of this study provide an unprecedented look at the actors involved in the dynamic changes of our protein balance and their involvement in pathophysiological states, which may guide the use of CRLs in the development of new therapies in the future.