Senescent cells form nuclear foci that contain the 26S proteasome.

The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.

Caspase cleaves Drosophila BubR1 to modulate spindle assembly checkpoint function and lifespan of the organism.

Caspases cleave over 1500 substrates in the human proteome in both lethal and non-lethal scenarios. However, reports of the physiological consequences of substrate cleavage are limited. Additionally, the manner in which caspase cleaves only a subset of substrates in the non-lethal scenario remains to be elucidated. BubR1, a spindle assembly checkpoint component, is a caspase substrate in humans, the physiological function of which remains unclear. Here, we found that caspases, especially Drice, cleave Drosophila BubR1 between the N-terminal KEN box motif and C-terminal kinase domain. By using proximity labelling, we found that Drice, but not Dcp-1, is in proximity to BubR1, suggesting that protein proximity facilitates substrate preference. The cleaved fragments displayed altered subcellular localization and protein-protein interactions. Flies that harboured cleavage-resistant BubR1 showed longer duration of BubR1 localization to the kinetochore upon colchicine treatment. Furthermore, these flies showed extended lifespan. Thus, we propose that the caspase-mediated cleavage of BubR1 limits spindle assembly checkpoint and organismal lifespan. Our results highlight the importance of the individual analysis of substrates in vivo to determine the biological significance of caspase-dependent non-lethal cellular processes.

Enhanced O-GlcNAcylation Mediates Cytoprotection under Proteasome Impairment by Promoting Proteasome Turnover in Cancer Cells.

The proteasome is a therapeutic target in cancer, but resistance to proteasome inhibitors often develops owing to the induction of compensatory pathways. Through a genome-wide siRNA screen combined with RNA sequencing analysis, we identified hexokinase and downstream O-GlcNAcylation as cell survival factors under proteasome impairment. The inhibition of O-GlcNAcylation synergistically induced massive cell death in combination with proteasome inhibition. We further demonstrated that O-GlcNAcylation was indispensable for maintaining proteasome activity by enhancing biogenesis as well as proteasome degradation in a manner independent of Nrf1, a well-known compensatory transcription factor that upregulates proteasome gene expression. Our results identify a pathway that maintains proteasome function under proteasome impairment, providing potential targets for cancer therapy.

Defective induction of the proteasome associated with T-cell receptor signaling underlies T-cell senescence.

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T-cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4+ T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4+ T cells upon T-cell receptor (TCR) stimulation underlie T-cell senescence. Proteasome dysfunction promotes senescence-associated phenotypes, including defective proliferation, cytokine production and increased levels of PD-1+ CD44High CD4+ T cells. Proteasome induction by TCR signaling via MEK-, IKK- and calcineurin-dependent pathways is attenuated with age and decreased in PD-1+ CD44High CD4+ T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4+ T-cell senescence.

FAM48A mediates compensatory autophagy induced by proteasome impairment.

Maintaining protein homeostasis is central to cell survival. The ubiquitin-proteasome system and autophagy play pivotal roles in protein quality control through protein degradation. Activities of these degradative pathways are carefully orchestrated, and autophagy is up-regulated during proteasome dysfunction for cellular homeostasis. However, the mechanism by which proteasome impairment induces compensatory autophagy has remained largely elusive. Here, we show that FAM48A mediates autophagy induction during proteasome inhibition. FAM48A is degraded by the proteasome and accumulates in cells by proteasome inhibition. Knockdown of FAM48A led to defective induction of autophagy during proteasome inhibition and accompanied by defective localization of Atg9 on recycling endosomes. Our results indicate that FAM48A is a kind of sensor that is required for compensatory autophagy induction upon proteasome impairment.

Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice.

The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α' subunit of casein kinase II (CK2α') as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

PAC1-PAC2 proteasome assembly chaperone retains the core α4-α7 assembly intermediates in the cytoplasm.

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α-rings and two ß-rings stacked in order of αßßα. The assembly of CP proceeds by ordered recruitment of ß-subunits on an α-ring with help of assembly chaperones PAC1-PAC2, PAC3-PAC4, and UMP1. However, the mechanism of α-ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α-ring assembly, which requires PAC3-PAC4. α1 and α3 can be incorporated independently into the core α4-α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1-PAC2 prevents nonproductive dimerization of α-ring assembly intermediates. We also found that PAC1-PAC2 overrides the effect of nuclear localization signals of α-subunits and retains α-ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α-ring and explain the mechanism by which CP assembly occurs in the cytoplasm.

Nuclear export of ubiquitinated proteins via the UBIN-POST system.

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

Ubiquitin-Binding Protein CG5445 Suppresses Aggregation and Cytotoxicity of Amyotrophic Lateral Sclerosis-Linked TDP-43 in Drosophila.

Ubiquitin-mediated protein degradation plays essential roles in proteostasis and is involved in the pathogenesis of neurodegenerative diseases in which ubiquitin-positive aberrant proteins accumulate. However, how such aberrant proteins are processed inside cells has not been fully explored. Here, we show that the product of CG5445, a previously uncharacterized Drosophila gene, prevents the accumulation of aggregate-prone ubiquitinated proteins. We found that ubiquitin conjugates were associated with CG5445, the knockdown of which caused the accumulation of detergent-insoluble ubiquitinated proteins. Furthermore, CG5445 rescued eye degeneration caused by the amyotrophic lateral sclerosis (ALS)-linked mutant TAR DNA-binding protein of 43 kDa (TDP-43), which often forms ubiquitin-positive aggregates in cells through the capacity of CG5445 to bind to ubiquitin chains. Biochemically, CG5445 inhibited the accumulation of insoluble forms and promoted their clearance. Our results demonstrate a new possible mechanism by which cells maintain ubiquitinated aggregation-prone proteins in a soluble form to decrease their cytotoxicity until they are degraded.

A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.

Fibrosis can disrupt tissue structure and integrity and impair organ function. Fibrosis is characterized by abnormal collagen accumulation in the extracellular matrix. Pharmacological inhibition of collagen secretion therefore represents a promising strategy for the management of fibrotic disorders, such as liver and lung fibrosis. Hsp47 is an endoplasmic reticulum (ER)-resident collagen-specific molecular chaperone essential for correct folding of procollagen in the ER. Genetic deletion of Hsp47 or inhibition of its interaction with procollagen interferes with procollagen triple helix production, which vastly reduces procollagen secretion from fibroblasts. Thus, Hsp47 could be a potential and promising target for the management of fibrosis. In this study, we screened small-molecule compounds that inhibit the interaction of Hsp47 with collagen from chemical libraries using surface plasmon resonance (BIAcore), and we found a molecule AK778 and its cleavage product Col003 competitively inhibited the interaction and caused the inhibition of collagen secretion by destabilizing the collagen triple helix. Structural information obtained with NMR analysis revealed that Col003 competitively binds to the collagen-binding site on Hsp47. We propose that these structural insights could provide a basis for designing more effective therapeutic drugs for managing fibrosis.

The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction.

In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes.

Sirt1-deficiency causes defective protein quality control.

Protein quality control is an important mechanism to maintain cellular homeostasis. Damaged proteins have to be restored or eliminated by degradation, which is mainly achieved by molecular chaperones and the ubiquitin-proteasome system. The NAD(+)-dependent deacetylase Sirt1 has been reported to play positive roles in the regulation of cellular homeostasis in response to various stresses. However, its contribution to protein quality control remains unexplored. Here we show that Sirt1 is involved in protein quality control in both an Hsp70-dependent and an Hsp70-independent manner. Loss of Sirt1 led to the accumulation of ubiquitinated proteins in cells and tissues, especially upon heat stress, without affecting proteasome activities. This was partly due to decreased basal expression of Hsp70. However, this accumulation was only partially alleviated by overexpression of Hsp70 or induction of Hsp70 upon heat shock in Sirt1-deficient cells and tissues. These results suggest that Sirt1 mediates both Hsp70-dependent and Hsp70-independent protein quality control. Our findings cast new light on understanding the role of Sirt1 in maintaining cellular homeostasis.

Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis.

Intracellular proteins tagged with ubiquitin chains are targeted to the 26S proteasome for degradation. The two subunits, Rpn10 and Rpn13, function as ubiquitin receptors of the proteasome. However, differences in roles between Rpn10 and Rpn13 in mammals remains to be understood. We analyzed mice deficient for Rpn13 and Rpn10. Liver-specific deletion of either Rpn10 or Rpn13 showed only modest impairment, but simultaneous loss of both caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates, which was recovered by re-expression of either Rpn10 or Rpn13. We also found that mHR23B and ubiquilin/Plic-1 and -4 failed to bind to the proteasome in the absence of both Rpn10 and Rpn13, suggesting that these two subunits are the main receptors for these UBL-UBA proteins that deliver ubiquitinated proteins to the proteasome. Our results indicate that Rpn13 mostly plays a redundant role with Rpn10 in recognition of ubiquitinated proteins and maintaining homeostasis in Mus musculus.

MKKS is a centrosome-shuttling protein degraded by disease-causing mutations via CHIP-mediated ubiquitination.

McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state.

Polyglutamine (polyQ)-expansion proteins cause neurodegenerative disorders including Huntington's disease, Kennedy's disease and various ataxias. The cytotoxicity of these proteins is associated with the formation of aggregates or other conformationally toxic species. Here, we show that the cytosolic chaperonin CCT (also known as TRiC) can alter the course of aggregation and cytotoxicity of huntingtin (Htt)-polyQ proteins in mammalian cells. Disruption of the CCT complex by RNAi-mediated knockdown enhanced Htt-polyQ aggregate formation and cellular toxicity. Analysis of the aggregation states of the Htt-polyQ proteins by fluorescence correlation spectroscopy revealed that CCT depletion results in the appearance of soluble Htt-polyQ aggregates. Similarly, overexpression of all eight subunits of CCT suppressed Htt aggregation and neuronal cell death. These results indicate that CCT has an essential role in protecting against the cytotoxicity of polyQ proteins by affecting the course of aggregation.