How cells decide on the life or death of a protein
In an international collaboration, researchers around David Haselbach at the IMP have investigated how the proteasome, the cell’s waste disposal machine, distinguishes between different molecular labels to decide a protein’s fate. By using cutting-edge cryo-electron microscopy and precisely engineered molecular labels, the team captured the first high-resolution view of the human proteasome as it reads different destruction signals. Their findings, published as a preprint on bioRxiv, shed light on how the proteasome makes its life-or-death decisions for cellular proteins.
Every eukaryotic cell needs a waste disposal system to stay healthy and function properly. At the heart of this system is the proteasome, a molecular machine that breaks down damaged or unneeded proteins, preventing their buildup and keeping the cell in balance.
The proteasome is guided in its cleanup job by ubiquitin—a molecular label that marks unwanted proteins for degradation, among other cellular functions. Ubiquitin tags typically form small protein chains, where individual ubiquitin molecules are linked together like a string of beads. These chains are then attached to one end of the target protein, indicating to the proteasome whether a protein should be destroyed, moved, or modified. By reading this tag, the proteasome determines the protein’s fate. This selective process is essential for a cell, regulating everything from cell division to stress responses. A malfunction of the proteasome can lead to severe diseases.
Scientists have long known that ubiquitin chains can be arranged in different shapes, each acting as a specific signal. But exactly how the proteasome reads and interprets these messages was not known.
One of the challenges lied in the visualisation of a complete ubiquitin chain bound to the proteasome. Protein breakdown happens extremely quickly, and when researchers attempt to observe the process, ubiquitin is often already gone. On top of that, ubiquitin chains are highly diverse and dynamic, making it difficult to capture them in the moment they interact with the proteasome.
Researchers from the lab of David Haselbach at the IMP with international collaborators now provided the first high-resolution view of the human proteasome as it reads different ubiquitin labels, revealing key insights into how the proteasome determines the life or death of a protein.
How the proteasome ‘feels out’ ubiquitin chains
Some ubiquitin chains form simple linear links, while others are branched, forming more complex tree-like structures. “The proteasome can distinguish different architectures to decide the protein’s fate. But we didn’t know how,” says David Haselbach, Technology
Platform Head for cryo-EM at the IMP.
The researchers focused on two types of ubiquitin chains: K48-linked chains, which serve as the primary signal for protein degradation, and K11/K48-branched chains, which play a key role in controlling the cell cycle. These two signals are crucial for different cellular processes, yet researchers still did not know why the proteasome treats them differently.
“To solve the challenge of visualising how these ubiquitin chains bind to the proteasome, we first had to create stable and uniform ubiquitin-tagged proteins,” says David Haselbach. “Ubiquitin chains are naturally variable, and for structural biology this makes it nearly impossible to capture clear images.” To obtain highly uniform samples required for structural analysis, the scientists teamed up with Kathrin Lang’s lab at ETH Zurich, which can build precise, identical ubiquitin chains.
The scientists then used single-particle cryo-electron microscopy, a technique that rapidly freezes purified proteins and captures thousands of images. These images were then used to build a detailed 3D model of the human proteasome interacting with the ubiquitin chains.
“It was surprising to see that ubiquitin chains don’t attach to the proteasome in a straight line, as was thought before.” says Sascha Amann, first author of the study and a student in the Vienna BioCenter PhD Program. “Instead, the main molecular tag that signals proteins for destruction wraps around a key proteasome component in a spiral shape, bringing important binding sites closer together.”
This explains why chains shorter than four ubiquitin molecules do not work as well for efficient protein breakdown—the right spacing is needed to properly activate the process. Previously, researchers thought the ubiquitin chain functioned more like a ruler, with fixed distances between each ubiquitin. In reality, the spiral shape brings the first and fourth ubiquitin molecules much closer together, allowing them to simultaneously engage two key ubiquitin receptors on the proteasome to kickstart the destruction process.
Meanwhile, K11/K48-branched chains, which play a major role in regulating the cell cycle, interact with entirely different regions of the proteasome “Different parts of the proteasome engage with different chain architectures, allowing it to tell these signals apart and determine the protein’s fate,” says Amann.
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AcceptThe researchers show that the proteasome ‘feels out’ the ubiquitin chain through multiple weak contact points spread across its surface, instead of relying on a single strong interaction. “This mechanism is important because it prevents the proteasome from grabbing onto the wrong proteins too tightly,” explains Haselbach. “A proofreading mechanism that ensures the proteasome’s high accuracy.”
The scientists have looked at the very first step of cellular waste disposal and validated their results in live cells. “Now we want to look at how other ubiquitin chain types drive protein breakdown, and how the proteasome actually commits to the process,” says David Haselbach.
These insights not only advance the understanding of cellular protein quality control, but could also have major biomedical applications, especially in drug development. By understanding how the proteasome distinguishes between different destruction signals, researchers could design more precise therapies to selectively remove disease-causing proteins.
Original Publication
Sascha J. Amann, Robert Kalis†, Maximilian Fottner†, Hannah Knaudt, Irina Grishkovskaya, Johannes Zuber, Kathrin Lang, Nicholas G. Brown, and David Haselbach#. "Structural basis for the ubiquitin chain recognition of the human 26S proteasome." bioRxiv.
#Corresponding author.
†Contributed equally.
Further reading