Research visualizes a precise mechanism allowing cells to sort their waste

For decades, the question of how proteins are labeled as defective or useless has remained open in the field of ubiquitin research. In a recent study, Brenda Schulman, director of the Max Planck Institute (MPI) for Biochemistry, and Gary Kleiger, chair of the Department of Chemistry and Biochemistry at the University of Las Vegas in Nevada, and their teams were able to visualize this precise mechanism, catalyzed by Cullin-RING Ligase E3, for the first time.

In an interview, Brenda Schulman explained what led them to their findings, as well as how this knowledge can be used to help treat diseases. The results of their study are published in the journal Nature Structural and molecular biology.

Cullin-RING ligases (CRLs) are complex nanomachines that are crucial for complex cell disposal and recycling systems. CRLs mark defective, toxic, or unnecessary proteins with repeating units of a small protein called ubiquitin, thereby forming a chain of ubiquitins in a process called poly-ubiquitin tagging.

In doing so, CRLs mark the protein as “to be degraded” and prevent toxic accumulation of unnecessary proteins. When this process fails, cellular waste can build up. Thus, mutations or dysfunctions altering the LCR are often associated with diseases, such as developmental disorders or cancers. Due to their key functions in maintaining the well-being of our cells, it is of fundamental importance to define and understand their molecular mechanisms.

Using a method called cryo-electron microscopy, or cryo-EM for short, it is now possible to visualize some of life’s smallest structures, such as CRLs, providing key insight into their functional states.

Brenda, you have dedicated half your life to research in the field of ubiquitin. Can you tell us exactly what fascinated you so much?

Ubiquitin is a fascinating protein, especially given its small size compared to most other proteins. Its name comes from “omnipresent” which means found everywhere. Indeed, we now know that ubiquitin is present almost everywhere: in plants, fungi, insects, animals and humans. And even though bacteria and viruses don’t produce ubiquitin, they hijack our ubiquitin to promote infections.

The action of ubiquitin is controlled by a system of hundreds of different molecular machines, called E3 ligases, which dictate where and when ubiquitin is attached to the cell’s waste products. Since ubiquitin is essentially ubiquitous, E3 ligases play an important role in activating and deactivating most cellular processes.

Studying ubiquitin for over 25 years now is like being a master detective in a long-running mystery series, especially since E3 ligases essentially pull the trigger on removing other proteins.

After solving one mystery, there is always another with even greater challenges. And the technologies in our forensic toolbox – for example CRISPR, chemical biology, and cryo-EM, to name a few – have become much more sophisticated. It therefore remains both an exciting and rewarding adventure to solve these mysteries with my colleagues.

Your recent study discovered how many different cellular proteins are marked very quickly by ubiquitin chains when they need to be removed. What led you and your team to this discovery?

Let me mention that this work is truly rewarding because it has remained a mystery for decades, and it’s a mystery I set out to solve when I started my first independent band 23 years ago.

Our collaborator, Gary Kleiger, of the University of Nevada, Las Vegas, showed that a CRL selects the target to mark, holds it in place while attaching poly-ubiquitin, and then releases the target so that the CRL can then tag another target for destruction. , all in a few milliseconds. In fact, this dizzying efficiency explains why until now it has not been possible to take photos of this process: it is too quick to capture with available techniques. We had to develop a completely new method to visualize how CRLs mark proteins for destruction.

Can you tell us more about the method you developed to reveal the mechanism?

We developed a chemical tool so that when CRL begins to tag a protein with poly-ubiquitin, the chemical acts as a trap, stopping the CRL at the key moment when the poly-ubiquitin tag is attached to the protein. This allowed us to visualize the complex using a structural biology method called cryo-EM.

Seeing all the molecular components come together led to the discovery of the CRL-catalyzed poly-ubiquitin labeling mechanism. Then, our long-time collaborator Gary Kleiger and his lab developed a new test to validate the model, which monitored these reactions in milliseconds: the first detected moment is 1/400th of a second!

What were the most promising observations that you believe will play a key role in the future development of the field?

Thanks to our chemical trap, we obtained different snapshots of CRL “in action”. These explained how polyubiquitin tagging works both on proteins naturally present in cells and on a protein when a drug-like molecule, MZ1, is applied. MZ1 triggers the elimination of a cancer-associated protein. All of these showed us many interesting facts, but there are three main conclusions.

The first was the revelation of the actual reaction mechanism of polyubiquitin labeling. Our structural snapshots showed all molecular components in synergy for ultra-rapid and specific catalysis. Indeed, speed and specificity are the distinctive characteristics of enzymes! This is a question that has been asked for over 30 years now and we are excited to be the first to show the mechanism of this incredibly rapid critical response.

The second key observation came from comparing the new data with our previously published work on how CRLs execute another, earlier step in marking proteins for removal. This earlier step involves the initial placement of a ubiquitin. The new study shows how the poly-ubiquitin tag, which contains several ubiquitins, is created. Crucial elements of the CRL molecular machine are radically reorganized during the first and last processes.

Perhaps most important is our finding that these features were observed for all CRLs we examined by tagging proteins with poly-ubiquitin tags, suggesting that our mechanism could apply to subsequent CRLs. in our cells.

What do you think your results will be used for in the future?

First of all, this is a big step forward in the field of ubiquitin, changing the way we think about a ubiquitous cellular system. We now know that all parts of the polyubiquitin-tagging machinery must come together in a specific way for the reaction to work with blazing speed and exquisite specificity.

Second, our study has implications for the emerging field of drug development called targeted protein degradation (TPD). Most TPD efforts use small molecules to recruit pathogenic proteins to CSF. This leads to polyubiquitin labeling and ultimately removal of the pathogenic protein. The new data indicate molecular and geometric constraints for the use of CRL for TPD, which we have shown for one of the pioneer degradative molecules, MZ1.

This area is very exciting and a complementary preprint on targeting CRLs via MZ1 has also been published on bioRxiv by the laboratory of Alessio Ciulli, who developed MZ1. Our findings could help develop new TPD drugs and understand how they work in diseased cells.