PA200-Mediated Proteasomal Protein Degradation and Regulation of Cellular Senescence.

Cellular senescence is closely related to DNA damage, proteasome inactivity, histone loss, epigenetic alterations, and tumorigenesis. The mammalian proteasome activator PA200 (also referred to as PSME4) or its yeast ortholog Blm10 promotes the acetylation-dependent degradation of the core histones during transcription, DNA repair, and spermatogenesis. According to recent studies, PA200 plays an important role in senescence, probably because of its role in promoting the degradation of the core histones. Loss of PA200 or Blm10 is a major cause of the decrease in proteasome activity during senescence. In this paper, recent research progress on the association of PA200 with cellular senescence is summarized, and the potential of PA200 to serve as a therapeutic target in age-related diseases is discussed.

Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (and Inactive) Proteasome Species.

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits ß2 and ß5 in the fully assembled proteasomes.

Proteasome activator Blm10 levels and autophagic degradation directly impact the proteasome landscape.

The proteasome selectively degrades proteins. It consists of a core particle (CP), which contains proteolytic active sites that can associate with different regulators to form various complexes. How these different complexes are regulated and affected by changing physiological conditions, however, remains poorly understood. In this study, we focused on the activator Blm10 and the regulatory particle (RP). In yeast, increased expression of Blm10 outcompeted RP for CP binding, which suggests that controlling the cellular levels of Blm10 can affect the relative amounts of RP-bound CP. While strong overexpression of BLM10 almost eliminated the presence of RP-CP complexes, the phenotypes this should induce were not observed. Our results show this was due to the induction of Blm10-CP autophagy under prolonged growth in YPD. Similarly, under conditions of endogenous BLM10 expression, Blm10 was degraded through autophagy as well. This suggests that reducing the levels of Blm10 allows for more CP-binding surfaces and the formation of RP-CP complexes under nutrient stress. This work provides important insights into maintaining the proteasome landscape and how protein expression levels affect proteasome function.

Proteasome Activator Blm10 Regulates Transcription Especially During Aging.

Background: Histones are basic elements of the chromatin and are critical to controlling chromatin structure and transcription. The proteasome activator PA200 promotes the acetylation-dependent proteasomal degradation of the core histones during spermatogenesis, DNA repair, transcription, and cellular aging and maintains the stability of histone marks. Objective: The study aimed to explore whether the yeast ortholog of PA200, Blm10, promotes degradation of the core histones during transcription and regulates transcription especially during aging. Methods: Protein degradation assays were performed to detect the role of Blm10 in histone degradation during transcription. mRNA profiles were compared in WT and mutant BY4741 or MDY510 yeast cells by RNA-sequencing. Results: The core histones can be degraded by the Blm10-proteasome in the non-replicating yeast, suggesting that Blm10 promotes the transcription-coupled degradation of the core histones. Blm10 preferentially regulates transcription in aged yeast, especially transcription of genes related to translation, amino acid metabolism, and carbohydrate metabolism. Mutations of Blm10 at F2125/N2126 in its putative acetyl-lysine binding region abolished the Blm10-mediated regulation of gene expression. Conclusion: Blm10 promotes degradation of the core histones during transcription and regulates transcription, especially during cellular aging, further supporting the critical role of PA200 in maintaining the stability of histone marks from the evolutionary view. These results should provide meaningful insights into the mechanisms underlying aging and the related diseases.

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington's disease (HD) research.

Transcriptional upregulation of proteasome activator Blm10 antagonizes cellular aging.

Cellular aging is associated with the damage to DNA, decline in proteasome activity, loss of histones and alteration of epigenetic marks. The atypical proteasome with the activator PA200 in mammals or its ortholog Blm10 in yeast promotes the acetylation-dependent degradation of the core histones during DNA repair or spermiogenesis. We show here that loss of PA200 or Blm10 is the leading cause of the decline in proteasome activity during aging, the latter of which conversely induces the transcription of Blm10. The transcription factor Crt1 suppressed, but the proteasome subunit Rpn4 promoted, the transcription of Blm10. On the contrary to deletion of Rpn4, deletion of Crt1 elevated Blm10 transcription upon DNA damage, reduced core histone levels during aging, and prolonged replicative lifespan. These results suggest that cells can antagonize aging by up-regulating transcription of Blm10, providing important insights into the mechanisms of aging and the aging-related diseases.

Nuclear Transport of Yeast Proteasomes.

Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.

To save or degrade: balancing proteasome homeostasis to maximize cell survival.

Autophagic degradation of proteasomes (termed proteaphagy) is a conserved mechanism by which cells eliminate excess or damaged particles. This clearance is induced rapidly when organisms are starved for nitrogen and, because proteasomes are highly abundant, their breakdown likely makes an important contribution to the amino acid pools necessary for survival. By contrast, our recent studies found that proteasomes are not degraded in response to carbon starvation, even though bulk macroautophagy is similarly activated. Instead, carbon starvation induces sequestration of proteasomes into membrane-less cytoplasmic condensates previously termed proteasome storage granules (PSGs), which protect proteasomes from autophagic degradation. Preserving proteasomes in PSGs enhances the ability of yeast cells to recover from a variety of stresses, implying that rapid remobilization of stored proteasomes when conditions improve is advantageous to cell fitness. Consequently, the choice of whether to save or degrade proteasomes can profoundly impact cell survival.

Nuclear Import of Yeast Proteasomes.

Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αß. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence.

Intracellular Dynamics of the Ubiquitin-Proteasome-System.

The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum ( ER) membranes. In prolonged quiescence, proteasome granules drop off the nuclear envelopeNE / ER membranes and migrate as droplet-like entitiesstable organelles  throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells, which comprise the majority of our body's cells.

Loss of a 20S proteasome activator in Saccharomyces cerevisiae downregulates genes important for genomic integrity, increases DNA damage, and selectively sensitizes cells to agents with diverse mechanisms of action.

Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of Blm10 did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10((-339aa)) localized to nuclei in untreated, nonproliferating (G(0)) cells, but not during G(1) S, G(2), and M. The results indicate Blm10 functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.