Search

A group of intrinsically disordered proteins (IDPs) are subject to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDPs/IDRs are targeted to the 20S proteasome via interaction with the C-terminus of the PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using a split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interactions with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

The Escherichia coli ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr 91st residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP 147th ) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe 143rd residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpY Y91F Q, ClpY Y91H Q, and ClpY Y91W Q, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced β-galactosidase activity of cpsB::lacZ , and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulA F143H , SulA F143K and SulA F143W , harboring a ring structure or a cation side-group in 143 rd residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpY Y91F Q and ClpY Y91H Q, were capable of effectively degrading SulA F143H and SulA F143K , highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π-π or cation-π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.

Since the discovery of ubiquitin conjugation as a cellular mechanism that triggers proteasomal degradation, the mode of substrate recognition by the ubiquitin-ligation system has been the holy grail of research in the field. This entails the discovery of recognition determinants within protein substrates, which are part of a degron, and explicit E3 ubiquitin (Ub)-protein ligases that trigger their degradation. Indeed, many protein substrates and their cognate E3′s have been discovered in the past 40 years. In the course of these studies, various degrons have been randomly identified, most of which are acquired through post-translational modification, typically, but not exclusively, protein phosphorylation. Nevertheless, acquired degrons cannot account for the vast diversity in cellular protein half-life times. Obviously, regulation of the proteome is largely determined by inherent degrons, that is, determinants integral to the protein structure. Inherent degrons are difficult to predict since they consist of diverse sequence and secondary structure features. Therefore, unbiased methods have been employed for their discovery. This review describes the history of degron discovery methods, including the development of high throughput screening methods, state of the art data acquisition and data analysis. Additionally, it summarizes major discoveries that led to the identification of cognate E3 ligases and hitherto unrecognized complexities of degron function. Finally, we discuss future perspectives and what still needs to be accomplished towards achieving the goal of understanding how the eukaryotic proteome is regulated via coordinated action of components of the ubiquitin-proteasome system. [ABSTRACT FROM AUTHOR]