Proteasome

Proteasomes are protein complexes that break down other proteins through proteolysis, a process that severs peptide bonds. The enzymes that drive these reactions are known as proteases. These complexes exist in all eukaryotes and archaea, as well as in certain bacteria. Within eukaryotic cells, proteasomes are found in both the nucleus and the cytoplasm. This degradation pathway is vital for numerous cellular functions, such as controlling the cell cycle, regulating gene expression, and managing oxidative stress.

The core of the complex is the 20S proteasome, a cylindrical structure made of four stacked rings that create a central pore. Each ring consists of seven individual proteins. The two inner rings are built from β subunits, which house between three and seven protease active sites inside the central chamber. Access to these proteases is controlled by a gate at the top of the 20S particle, which is regulated by larger protein complexes, including the 19S Regulatory Particle. Together, the 20S core and the 19S particle form the 26S proteasome. In eukaryotes, proteins marked with a small tag called ubiquitin are directed to the 26S proteasome, representing the final step of the ubiquitin-proteasome system (UPS). This system is a primary mechanism cells use to control the concentration of specific proteins and to dispose of misfolded ones.

For a protein to be degraded by the 26S proteasome, it needs two things: a ubiquitin tag and an unstructured region roughly 25 amino acids long. If a protein lacks this unstructured region, a motor protein—cdc48 in yeast or p97 in humans—can generate it. This motor, along with its cofactors Npl4 and Ufd1, unfolds the ubiquitin tag to create the necessary unstructured stretch. The tagging process itself involves a cascade of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Once a single ubiquitin is attached, it signals other ligases to add more ubiquitin molecules, forming a polyubiquitin chain. The proteasome binds this chain and, using ATP, degrades the tagged protein. This degradation produces peptides about seven to eight amino acids long, which can be broken down further into shorter sequences and reused to build new proteins.

Before the discovery of the ubiquitin-proteasome system, scientists believed that lysosomes—membrane-bound organelles filled with acidic proteases—were the main site for protein degradation, handling external proteins and damaged organelles. However, in 1977, Joseph Etlinger and Alfred L. Goldberg found that ATP-dependent protein degradation occurred in reticulocytes, which lack lysosomes, hinting at a second degradation pathway. The following year, this pathway was shown to involve several distinct protein chains, a novel feature for proteases at the time. Later research on histone modifications revealed an unexpected covalent bond between a lysine side chain on histones and the C-terminal glycine of ubiquitin, a protein with no known function. It then emerged that a protein previously linked to proteolytic degradation, called ATP-dependent proteolysis factor 1 (APF-1), was actually ubiquitin. The proteolytic activities of this system were isolated as a multi-protein complex, initially named the multi-catalytic proteinase complex by Sherwin Wilk and Marion Orlowski. Eventually, the ATP-dependent complex responsible for ubiquitin-triggered degradation was identified and called the 26S proteasome.

Much of the foundational work on this system took place in the late 1970s and early 1980s at the Technion, in Avram Hershko’s lab, where Aaron Ciechanover was a graduate student. A year-long sabbatical by Hershko in Irwin Rose’s lab at the Fox Chase Cancer Center provided key conceptual breakthroughs, though Rose later minimized his own role. The three shared the 2004 Nobel Prize in Chemistry for their discovery. While electron microscopy (EM) data from the mid-1980s showed the stacked-ring structure of the proteasome, the first X-ray crystallography structure of the core particle wasn’t solved until 1994. Wolfgang Baumeister’s group later used cryo-EM to reveal the overall architecture of the 26S proteasome, enabling biochemical experiments that outlined a general mechanism for ubiquitin-dependent degradation. In 2018, the first structure of the yeast 26S proteasome, followed by the first atomic structures of the human 26S proteasome holoenzyme bound to a polyubiquitylated substrate, were solved by cryogenic electron microscopy. These structures confirmed how the substrate is recognized, deubiquitylated, unfolded, and degraded. Detailed biochemistry has since established a general mechanism: the substrate binds to the proteasome, an unstructured region engages the AAA motor, triggering a major conformational change, then deubiquitination by Rpn11 occurs during translocation, followed by unfolding and proteolysis within the 20S core particle.

Cryo-electron tomography (cryo-ET) has also provided unique views of proteasomes inside cells. In neurons, proteasomes were found to exist in the same ground-state and processing conformations.