Formation of aggresomes with hydrogel-like characteristics by proteasome inhibition.

The spatiotemporal sequestration of misfolded proteins is a mechanism by which cells counterbalance proteome homeostasis upon exposure to various stress stimuli. Chronic inhibition of proteasomes results in a large, juxtanuclear, membrane-less inclusion, known as the aggresome. Although the molecular mechanisms driving its formation, clearance, and pathophysiological implications are continuously being uncovered, the biophysical aspects of aggresomes remain largely uncharacterized. Using fluorescence recovery after photobleaching and liquid droplet disruption assays, we found that the aggresomes are a homogeneously blended condensates with liquid-like properties similar to droplets formed via liquid-liquid phase separation. However, unlike fluidic liquid droplets, aggresomes have more viscosity and hydrogel-like characteristics. We also observed that the inhibition of aggresome formation using microtubule-disrupting agents resulted in less soluble and smaller cytoplasmic speckles, which was associated with marked cytotoxicity. Therefore, the aggresome appears to be cytoprotective and serves as a temporal reservoir for dysfunctional proteasomes and substrates that need to be degraded. Our results suggest that the aggresome assembles through distinct and potentially sequential processes of energy-dependent retrograde transportation and spontaneous condensation into a hydrogel.

ECPAS/Ecm29-mediated 26S proteasome disassembly is an adaptive response to glucose starvation.

The 26S proteasome comprises 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in cells exist as free 20S complexes; however, our mechanistic understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation uncouples 26S holoenzymes into 20S and 19S subcomplexes. Subcomplex affinity purification and quantitative mass spectrometry reveal that Ecm29 proteasome adaptor and scaffold (ECPAS) mediates this structural remodeling. The loss of ECPAS abrogates 26S dissociation, reducing degradation of 20S proteasome substrates, including puromycylated polypeptides. In silico modeling suggests that ECPAS conformational changes commence the disassembly process. ECPAS is also essential for endoplasmic reticulum stress response and cell survival during glucose starvation. In vivo xenograft model analysis reveals elevated 20S proteasome levels in glucose-deprived tumors. Our results demonstrate that the 20S-19S disassembly is a mechanism adapting global proteolysis to physiological needs and countering proteotoxic stress.

Effects of mTORC1 inhibition on proteasome activity and levels.

The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance. [BMB Reports 2022; 55(4): 161-165].

Evaluation of Immunoproteasome-Specific Proteolytic Activity Using Fluorogenic Peptide Substrates.

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (ß1i/PSMB9, ß2i/PSMB10, and ß5i/PSMB8) instead of constitutively expressed counterparts (ß1/PSMB6, ß2/PSMB7, and ß5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining ß5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

CHIP-mediated hyperubiquitylation of tau promotes its self-assembly into the insoluble tau filaments.

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3ß and CHIP, respectively. The resulting phospho-ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one-three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho-ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho-ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a "multiple-hit model," where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.