The proteasome is a protein complex responsible for the degradation of intracellular proteins, playing a central role in protein homeostasis by regulating various cellular processes. Intracellular proteins are labeled with ubiquitin molecules and degraded by the 26S system. Oxidized proteins can be degraded in an ATP- and poly-ubiquitin-independent manner by the free 20S proteasome - the catalytic core of the proteasome dissociated from regulatory units.

However, as evidence of the free 20S proteasome activity is predominantly based on in vitro demonstrations, there is still controversy about its physiological role in cells. The scientific literature on the subject is vast and divided. In a review article published in the journal BBA General Subjects, scientists from the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma), Marilene Demasi, at Instituto Butantan, and Fernanda Marques da Cunha, at Universidade Federal de São Paulo, propose a critical analysis of the available experimental data on the physiological role of the free 20S proteasome.

"Our conclusion is that there is no unequivocal evidence of ubiquitin-independent proteolysis through the free 20S proteasome, which must still be demonstrated in vivo. It’s still an open field. But the data accumulated to date challenges the notion that all proteasomal degradation implies the polyubiquitylation of substrate and the 26S proteasome is the only player in degradation," Demasi said.

According to the researcher, about 80% of the intracellular proteins are degraded by the proteasome, which regulates several cellular processes such as metabolism, signaling, cell division, and cell death. The Nobel Prize in Chemistry 2004 was awarded to three biochemists for the discovery of ubiquitin-mediated protein degradation. Currently, proteasome inhibitors are used as antitumor therapy. Proteasome also plays an important role in adaptive immune responses and in adaption to oxidative stress.

Free 20S proteasome

The proteasome is a ubiquitous and highly plastic protease conserved in all eukaryotes. It is composed of a cylindrical-shaped catalytic unit, called 20S that contains the catalytic sites. Regulatory units are coupled to one or both sides of the 20S core particle, with the 19S unit being the most abundant. When coupled to the 19S regulatory unit, the proteasome is named 26S. Protein substrates are mostly recognized through the poly-ubiquitin chain by the 19S regulatory unit. Once attached to the 19S subunit, the substrate is deubiquitylated, unfolded and translocated to the catalytic chamber. All these enzymatic processes consume energy in the form of ATP.

When the 20S is free, that is, uncoupled, it will degrade proteins by ATP- and poly-ubiquitin-independent mechanisms, which have not yet been well understood.

One hypothesis is that oxidation leads to chemical modifications to proteins that result in conformational changes, with exposure of hydrophobic residues. These hydrophobic patches are recognized by the unbound 20S proteasome without the need for ubiquitin targeting.

According to the researchers, two examples of indirect evidence of the free 20S proteasome activity are the in vitro experiments in which it mediates proteolysis, and demonstrations of an intracellular process of protein degradation independently of poly-ubiquitylation.

"A direct and definitive demonstration is very difficult to obtain because protein degradation without regulatory units happens in the absence of ATP and it is impossible to have cells completely depleted of ATP,” explains Cunha.

In the review, the researchers cited works that have been trying to calculate how much free 20S protease exists inside a cell. Using electron cryotomography of intact cells, scientists were able to identify the 20S particle capped at one or both ends with a 19S regulatory unit. They found that only approximately 25% of the detected proteasome pool was formed by 19S-20S-19S complex and that the remaining 75% of the pool was represented by the 20S proteasome capped at only one end. For the researchers, this could suggest the possibility of a bi-functional proteasome that would allow protein degradation via ATP- and ubiquitin-dependent or -independent pathways. The presence of the 20S proteasome completely uncapped was not determined in that study.

The free 20S proteasome has only recently been shown, for the first time, embedded into neuronal membranes generating peptides to the extracellular neuronal environment. This is a clear example of a physiological role of the free 20S proteasome.

An important issue to be addressed when considering proteolysis by the free 20S proteasome in vivo is whether there are mechanisms to promote the opening of the 20SPT gate in the absence of regulatory units. When not coupled, the 20S particle is closed; it opens when coupled to unit 19S. In 2012, Demasi team showed that the gate opening of the 20S particle in the yeast Saccharomyces cerevisiae take place through the in vivo S-glutathionylation, a redox post-translational modification, of specific cysteine ​​residues. One of the residues involved is conserved throughout yeast to humans.

The team recently performed the mutation of those specific cysteines, and the results obtained so far confirm the importance of those residues for the structure of the protease complex. By correlating the yeast chronological lifespan with the frequency with which the free 20S proteasome is found in the open or closed state, the researchers found that there is a 25% increase in lifespan when the proteasome is mostly open. According to Demasi, these results, which will be published soon, corroborate the physiological role of the free 20S proteasome, although they are still indirect evidence.

Also aiming to assess the physiological function of the proteasome, the researcher Fernanda Cunha investigates how the caloric restriction in yeast affects the activity of the proteasome. Results published in 2011 show that yeast cells cultured in caloric restriction have increased proteasome activity without an increase in the number of proteasome particles, indicating a possible structural modification. The group is now searching for post-translational modifications induced by calorie restrinction in the 20S proteasome that could be responsible for the increased activity.

The researchers believe that new techniques, such as electron cryotomography (cryo-ET) and other powerful imaging tools which allow the observation of molecular structures in live cells such as super-resolution microscopy, will facilitate the investigation of the role played by the free 20S proteasome in cells.

The review article The physiological role of the free 20S proteasome in protein degradation: A critical review, by Marilene Demasi and Fernanda Marques da Cunha, can be read by subscribers here.