ubiread: Cracking of the ubiquitin code of proteolysis

Ubiquitin marks proteins for degradation. This allows ubiquitin molecules to be bound by different types and numbers of different chains. Researchers at the Max Planck Institute of Biochemistry (MPIB) have developed a new Ubiread technology to decipher various combinations of ubiquitin molecules (ubiquitin codes).

Using Ubiread, scientists label fluorescent proteins with specific ubiquitin codes to track intracellular degradation. This study, published in molecular cells, revealed which ubiquitin codes can or cannot induce intracellular proteolysis.

Proteins are components of life and maintain cell structure and function. However, if proteins become damaged, accidentally or outdated, they can lead to a variety of diseases, ranging from Alzheimer’s and Parkinson’s to cancer and muscular dystrophy. To prevent this, we have developed a sophisticated system that marks unnecessary proteins because cells are degraded by small proteins called ubiquitin.

Ubiquitin is a cellular tag that tells you what to do with that protein. The ubiquitin code can be a single ubiquitin tag or multiple ubiquitins attached together.

Leo Kiss, the first author of the molecular machinery in the department of Brenda Schulman, and the postdoctoral and MPIB signaling, “The ubiquitin code is really appealing due to its complexity. Linking one ubiquitin molecule in another way happens in eight different ways.

“And as if it wasn’t complicated enough, there are many different types of chains that can fork from existing chains. There are so many possibilities that we still don’t fully understand the information these structures encode. That’s where Ubiread, our technology, comes in.

The complexity of the ubiquitin code poses a major challenge for researchers studying protein degradation. The various combinations of chain types, lengths and shapes create vast strips of cords that remain difficult to decipher.

One of the biggest hurdles is the lack of a systematic approach to observing cell proteolysis. There are limitations to the current method. Cell-based approaches cannot create specific ubiquitin chains in cells on demand, but biochemical techniques often fail to replicate the degradation behavior seen in cells. This has resulted in conflicting results in various studies.

Ubiread Technology

To unravel this ubiquitin code and to better understand protein degradation, Kiss and Brenda Schulman have developed Ubiread (reporter evaluation after ubiquitinated reporter evaluation) in collaboration with Leojames, director of Host Pathogenic Biology, a research group at the MRC Institute for Molecular Biology in Cambridge, UK.

This new approach allows us to study the cellular degradation of proteins carrying defined ubiquitin codes. Researchers are tagged with fluorescent proteins with known ubiquitin codes. The tagged fluorescent protein is then delivered to the cells. The fate of the protein can be followed based on the fluorescence intensity correlated with the amount of protein. When recycled, fluorescence is lost.

Using Ubiread, researchers found that intracellular degradation was faster than the degradation of the same substrate in vitro with purified degradation mechanisms. It only takes one minute to break down half of the protein tagged in the cellular environment. Furthermore, it can be shown that proteins tagged with a certain type of ubiquitin chain, called K48, are rapidly degraded, and that proteins tagged with K63 quickly lose their tags and avoid recycling.

The researchers also compared the lengths of different ubiquitin chains. Kiss said, “A surprising thing is that three ubiquitin molecules were already sufficient to effectively recycle fluorescent proteins. However, if these ubiquitins do not form directly on the substrate and are formed on another ubiquitin chain, this code can be lost. The exact context is important.”

Schulman, director of MPIB, said, “Our findings underscore the importance of investigating ubiquitin chains and their functions in native cellular environments. We have shown that Ubiread is a versatile tool for analyzing different types of proteins that fall within the context of proteins like ubiquitin, from the context of proteins like ubiquitin or ubiquitin. More insights into complex ubiquitin systems.”