Cracking the Code of Protein Degradation

Scientists from the Max-Planck-Institute of Biochemistry developed a new technology to decipher the intricacies of the protein degradation code within cells.

Ubiquitin marks proteins for degradation, whereby ubiquitin molecules can be combined in different types and numbers forming different chains. Researchers at the Max Planck Institute of Biochemistry (MPIB) have developed the new UbiREAD technology to decode the various combinations of ubiquitin molecules – the ubiquitin code - which determine how proteins are degraded in cells. Using UbiREAD, scientists label fluorescent proteins with specific ubiquitin codes and track their degradation in cells. The study, published in Molecular Cell, revealed which Ubiquitin code can or cannot induce intracellular protein degradation. It also showed that protein degradation in cells is faster than in “test tubes”, and found a molecular hierarchy in more complicated ubiquitin tags.

Proteins are the building blocks of life, maintaining cellular structure and function. However, when proteins become damaged, misfolded, or obsolete, they can lead to a range of diseases, from Alzheimer’s and Parkinson’s to cancer and muscular dystrophy. To prevent this, cells have developed a sophisticated system to mark unwanted proteins for degradation with a small protein called ubiquitin.

Ubiquitin chains

Ubiquitin is a cellular tag that marks what should happen to that protein. The ubiquitin code can be a single ubiquitin tag or multiple ubiquitins attached together. Leo Kiss, first author and Postdoc in Brenda Schulman’s department Molecular Machines and Signaling at the MPIB explains: “The ubiquitin code is truly fascinating due to its complexity: Linking one ubiquitin molecule to another can happen in 8 different ways. These attachments can repeat multiple times, creating chains of varying lengths. And as if that weren’t complex enough, different types of chains can branch off from existing ones. With so many possibilities, we do not yet fully understand the information these structures encode. That’s where our technology, UbiREAD, comes in – it acts like a scanner that can decode what these different ubiquitin chains do inside cells.”

How to analyze the ubiquitin code

The intricacy of the ubiquitin code poses a significant challenge for researchers studying protein degradation. The various combinations of chain types, lengths, and shapes result in a vast swath of codes that have remained difficult to decipher. One of the biggest hurdles has been the lack of a systematic approach to observe protein degradation in cells. Current methods have limitations: while cell-based approaches can't create specific ubiquitin chains in cells on demand, biochemical methods often fail to replicate the degradation behaviors seen in cells. This has led to conflicting results across different studies.

UbiREAD technology

To unravel this ubiquitin code and understand protein degradation better, Leo Kiss and Brenda Schulman in collaboration with Leo James, head of the research group “Host-Pathogen Biology”, at the MRC Laboratory of Molecular Biology in Cambridge, UK have developed UbiREAD (Ubiquitinated Reporter Evaluation After intracellular Delivery). This new approach allows studying cellular degradation of proteins carrying a defined ubiquitin code. The researchers tag a fluorescent protein with a known ubiquitin code. Then, they deliver the tagged fluorescent protein to the cells. On the basis of the fluorescence intensity, which correlates with the amount of protein, they can follow the fate of the protein: If it is still intact fluorescence remains, if it is recycled fluorescence is lost.

New insights

Using UbiREAD, researchers discovered that intracellular degradation is faster than the degradation of the same substrate in a test tube with purified degradation machinery. It takes only a minute to degrade half of the tagged protein in cellular environment. Additionally, they could show that proteins tagged with a specific type of ubiquitin chain, called K48, are rapidly degraded, while proteins tagged with K63, quickly lose their tag and avoid recycling.

The researchers also compared different ubiquitin chain lengths. Leo Kiss says: “Surprisingly, already three ubiquitin molecules were sufficient to effectively recycle the fluorescent protein. However, this code can be lost when these ubiquitins are not formed on the substrate directly, but on another ubiquitin chain. The exact context matters.”

Brenda Schulman, Director at the MPIB, summarizes: “Our findings highlight the importance of investigating ubiquitin chains and their functions in their native cellular environment. We have shown, that UbiREAD is a versatile tool to analyze intracellular degradation of proteins conjugated to various types of ubiquitin or ubiquitin like protein chains in different cellular contexts. In the near future, we will continue to use the method to gain many more insights into the complex ubiquitin system.”

 

Glossary
Homeostasis: Cellular homeostasis is the maintenance of a stable internal state in cells despite external changes. It includes the regulation of proteins, pH, ion concentration and energy balance. Complex signaling pathways control these processes to prevent cell damage. Disruptions can lead to disease.

Protein degradation: is the process by which cells break down proteins into smaller peptides and amino acids, maintaining protein homeostasis and regulating cellular functions. This process involves two main pathways: the ubiquitin-proteasome system for intracellular proteins and the lysosomal proteolysis pathway for extracellular proteins and cell-surface receptors.

UbiREAD: abbreviation for Ubiquitinated Reporter Evaluation After intracellular Delivery. It is a new technology, that allows researchers to study degradation of proteins with defined ubiquitin tags inside of mammalian cells.

Ubiquitin: Ubiquitin is a small protein that is often attached to other proteins as a marker. This process is called ubiquitylation.

Ubiquitin-proteasome system (UPS): is a cellular pathway that tags proteins with ubiquitin molecules, marking them for degradation by the proteasome, a large protease complex. This system is crucial for maintaining cellular homeostasis by removing damaged or unnecessary proteins and regulating various cellular functions, such as cell cycle progression and stress responses.