Systematic identification of 20S proteasome substrates.

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

Allosteric regulation of the 20S proteasome by the Catalytic Core Regulators (CCRs) family.

Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 ß-subunit with attenuation of the complex's three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome's enzymatic subunits but rather propagates through the non-catalytic subunit PSMB4. This way of 20S proteasome-specific attenuation opens avenues for decoupling the 20S and 26S proteasome degradation pathways as well as for developing selective 20S proteasome inhibitors.

Regulation of the 20S Proteasome by a Novel Family of Inhibitory Proteins.

Aims: The protein degradation machinery plays a critical role in the maintenance of cellular homeostasis, preventing the accumulation of damaged or misfolded proteins and controlling the levels of regulatory proteins. The 20S proteasome degradation machinery, which predominates during oxidative stress, is able to cleave any protein with a partially unfolded region, however, uncontrolled degradation of the myriad of potential substrates is improbable. This study aimed to identify and characterize the regulatory mechanism that controls 20S proteasome-mediated degradation. Results: Using a bioinformatic screen based on known 20S proteasome regulators, we have discovered a novel family of 20S proteasome regulators, named catalytic core regulators (CCRs). These regulators share structural and sequence similarities, and coordinate the function of the 20S proteasome by affecting the degradation of substrates. The CCRs are involved in the oxidative stress response via Nrf2, organizing into a feed-forward loop regulatory circuit, with some members stabilizing Nrf2, others being induced by Nrf2, and all of them inhibiting the 20S proteasome. Innovation and Conclusion: These data uncover a new family of regulatory proteins that utilize a fine-tuned mechanism to carefully modulate the activity of the 20S proteasome, in particular under conditions of oxidative stress, ensuring its proper functioning by controlling the degradative flux.

CSNAP, the smallest CSN subunit, modulates proteostasis through cullin-RING ubiquitin ligases.

The cullin-RING ubiquitin E3 ligase (CRL) family consists of ~250 complexes that catalyze ubiquitylation of proteins to achieve cellular regulation. All CRLs are inhibited by the COP9 signalosome complex (CSN) through both enzymatic (deneddylation) and nonenzymatic (steric) mechanisms. The relative contribution of these two mechanisms is unclear. Here, we decouple the mechanisms using CSNAP, the recently discovered ninth subunit of the CSN. We find that CSNAP reduces the affinity of CSN toward CRL complexes. Removing CSNAP does not affect deneddylation, but leads to global effects on the CRL, causing altered reproductive capacity, suppressed DNA damage response, and delayed cell cycle progression. Thus, although CSNAP is only 2% of the CSN mass, it plays a critical role in the steric regulation of CRLs by the CSN.

Post-translational regulation of p53 function through 20S proteasome-mediated cleavage.

The tumor suppressor p53 is a transcription factor that regulates the expression of a range of target genes in response to cellular stress. Adding to the complexity of understanding its cellular function is that in addition to the full-length protein, several p53 isoforms are produced in humans, harboring diverse expression patterns and functionalities. One isoform, Δ40p53, which lacks the first transactivation domain including the binding region for the negative regulator MDM2, was shown to be a product of alternative translation initiation. Here we report the discovery of an alternative cellular mechanism for Δ40p53 formation. We show that the 20S proteasome specifically cleaves the full-length protein (FLp53) to generate the Δ40p53 isoform. Moreover, we demonstrate that a dimer of FLp53 interacts with a Δ40p53 dimer, creating a functional hetero-tetramer. Consequently, the co-expression of both isoforms attenuates the transcriptional activity of FLp53 in a dominant negative manner. Finally, we demonstrate that following oxidative stress, at the time when the 20S proteasome becomes the major degradation machinery and FLp53 is activated, the formation of Δ40p53 is enhanced, creating a negative feedback loop that balances FLp53 activation. Overall, our results suggest that Δ40p53 can be generated by a 20S proteasome-mediated post-translational mechanism so as to control p53 function. More generally, the discovery of a specific cleavage function for the 20S proteasome may represent a more general cellular regulatory mechanism to produce proteins with distinct functional properties.

The Parkinson's-associated protein DJ-1 regulates the 20S proteasome.

The Parkinson's-associated protein, DJ-1, is a highly conserved homodimer, ubiquitously expressed in cells. Here we demonstrate that DJ-1 is a 20S proteasome regulator. We show that DJ-1 physically binds the 20S proteasome and inhibits its activity, rescuing partially unfolded proteins from degradation. Consequently, DJ-1 stabilizes the cellular levels of 20S proteasome substrates, as we show for α-synuclein and p53. Furthermore, we demonstrate that following oxidative stress, DJ-1 is involved in the Nrf2-dependent oxidative stress response that leads to the upregulation of both the 20S proteasome and its regulator, NQO1. Overall, our results suggest a regulatory circuit in which DJ-1, under conditions of oxidative stress, both upregulates and inhibits the 20S proteasome, providing a rigorous control mechanism at a time when the 20S proteasome becomes the major proteolytic machinery. Such a tight regulation of the 20S proteasome may sustain the balance between the need to rapidly eliminate oxidatively damaged proteins and maintain the abundance of native, intrinsically unstructured proteins, which coordinate regulatory and signalling events.