Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Abstract

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex-the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

dagLogo: An R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data

“…To facilitate the discovery of conserved patterns from increasingly accumulated proteomic data, we developed the dagLogo package, which have been well downloaded and cited by researchers [4,80,91,92]. Though it does not provide a graphic user interface as other web applications for sequence logo generators (See S1 Table), our package helps to identify and visualize differentially used AA residues or AA residue groups in given protein sequences, improving and extending the sophisticated logo generator iceLogo.…”

Structural Insights Into Substrate Recognition and Processing by the 20S Proteasome

“…Since ubiquitinated substrates are preferentially degraded by 26S proteasomes, it is reasonable to assume that the non-ubiquitinated IDPs would be degraded by 20S proteasomes. Indeed, in certain in vitro experiments, the rate of proteolysis of non-ubiquitinated disordered proteins by 20S proteasomes was faster than by 26S proteasomes [59]. This could be explained by the hindrances to substrate processing and translocation found in the resting state of 26S proteasomes (Figure 2), nevertheless, the question remains how are disordered proteins be targeted to 20S proteasomes and what activates these proteases for proteolysis?…”

“…Preferred substrates for degradation by 20S proteasomes are unstructured, unfolded or misfolded proteins. Various studies have addressed the ability of 20S proteasomes to degrade proteins of this category both in in vitro and in vivo conditions [9,25,26,28,57,59,61,[78][79][80][81]. Hence, the targeting signal to 20S proteasomes is considered to be an unstructured polypeptide segment on a substrate.…”

“…Lacking ubiquitin-binding domains or ATPase subunits, 20S proteasome activity is adversely affected by the presence of globular domains within a partially unstructured polypeptide, or by conjugation of a substrate to the tightly folded ubiquitin. A recent study demonstrated that addition of ubiquitin units to a model disordered substrate, CyclinB1, slowed down the overall degradation rate of CyclinB1 by free 20S proteasomes [59]. Under such circumstances, 20S proteasomes either release the globular domain [11] or degrade the globular domain along with the unstructured portion of the substrate [59].…”

“…Although its functionality as an independently active enzyme under cellular conditions is a matter of debate, recent advances have highlighted a role for 20S as a functional proteasome in cells. Various reports suggest a role of 20S as an emergency proteasome under cellular stress, for example oxidative stress or hypoxic stress [25,54,[57][58][59][60][61], to provide survival benefits under proteotoxicity [26].…”