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BMB Reports Apr 2022The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell...
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].
Topics: Autophagy; Autophagy-Related Protein-1 Homolog; Humans; Mechanistic Target of Rapamycin Complex 1; Proteasome Endopeptidase Complex; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 35321785
DOI: 10.5483/BMBRep.2022.55.4.032 -
The Journal of Biological Chemistry Nov 2022The REGγ-20S proteasome is an ubiquitin- and ATP-independent degradation system, targeting selective substrates, possibly helping to regulate aging. The studies we...
The REGγ-20S proteasome is an ubiquitin- and ATP-independent degradation system, targeting selective substrates, possibly helping to regulate aging. The studies we report here demonstrate that aging-associated REGγ decline predisposes to decreasing tau turnover, as in a tauopathy. The REGγ proteasome promotes degradation of human and mouse tau, notably phosphorylated tau and toxic tau oligomers that shuttle between the cytoplasm and nuclei. REGγ-mediated proteasomal degradation of tau was validated in 3- to 12-month-old REGγ KO mice, REGγ KO;PS19 mice, and PS19 mice with forebrain conditional neuron-specific overexpression of REGγ (REGγ OE) and behavioral abnormalities. Coupled with tau accumulation, we found with REGγ-deficiency, neuron loss, dendrite reduction, tau filament accumulation, and microglial activation are much more prominent in the REGγ KO;PS19 than the PS19 model. Moreover, we observed that the degenerative neuronal lesions and aberrant behaviors were alleviated in REGγ OE;PS19 mice. Memory and other behavior analysis substantiate the role of REGγ in prevention of tauopathy-like symptoms. In addition, we investigated the potential mechanism underlying aging-related REGγ decline. This study provides valuable insights into the novel regulatory mechanisms and potential therapeutic targets for tau-related neurodegenerative diseases.
Topics: Humans; Animals; Mice; Infant; Proteasome Endopeptidase Complex; Tauopathies; Autoantigens; Cytoplasm; Aging
PubMed: 36209822
DOI: 10.1016/j.jbc.2022.102571 -
Biomolecules Aug 2020Protein homeostasis (proteostasis) is essential for the cell and is maintained by a highly conserved protein quality control (PQC) system, which triages newly... (Review)
Review
Protein homeostasis (proteostasis) is essential for the cell and is maintained by a highly conserved protein quality control (PQC) system, which triages newly synthesized, mislocalized and misfolded proteins. The ubiquitin-proteasome system (UPS), molecular chaperones, and co-chaperones are vital PQC elements that work together to facilitate degradation of misfolded and toxic protein species through the 26S proteasome. However, the underlying mechanisms are complex and remain partly unclear. Here, we provide an overview of the current knowledge on the co-chaperones that directly take part in targeting and delivery of PQC substrates for degradation. While J-domain proteins (JDPs) target substrates for the heat shock protein 70 (HSP70) chaperones, nucleotide-exchange factors (NEFs) deliver HSP70-bound substrates to the proteasome. So far, three NEFs have been established in proteasomal delivery: HSP110 and the ubiquitin-like (UBL) domain proteins BAG-1 and BAG-6, the latter acting as a chaperone itself and carrying its substrates directly to the proteasome. A better understanding of the individual delivery pathways will improve our ability to regulate the triage, and thus regulate the fate of aberrant proteins involved in cell stress and disease, examples of which are given throughout the review.
Topics: Animals; HSP70 Heat-Shock Proteins; Humans; Proteasome Endopeptidase Complex; Protein Folding; Proteostasis
PubMed: 32759676
DOI: 10.3390/biom10081141 -
Blood Transfusion = Trasfusione Del... Jan 2022Proteasomes are proteolytic complexes with prominent roles in the control of protein homeostasis and cellular viability. However, little is known about the effects of...
BACKGROUND
Proteasomes are proteolytic complexes with prominent roles in the control of protein homeostasis and cellular viability. However, little is known about the effects of storage and glucose-6-phosphate dehydrogenase deficiency (G6PD) on the activity and topology of red blood cell (RBC) proteasomes.
MATERIALS AND METHODS
We investigated the concentration (by GeLC-MS proteomics analysis and immunoblotting), activity (by using peptide substrates and proteasome inhibitors), and subcellular/extracellular distribution (following cell fractionation and isolation of extracellular vesicles, respectively) of RBC proteasomes in fresh blood and RBCs from control and G6PD donors following storage in leukoreduced units. RBC proteasome activity was also tested in transfusion-mimicking conditions in vitro.
RESULTS
Stored RBCs were characterised by decreased cytosolic proteasome activity compared to fresh RBCs but increased membrane activity and protein concentration levels. Active proteasomes along with other "repair or destroy" proteins are recruited to the membrane during storage. A proportion of them is released in the supernatant in soluble form or inside extracellular vesicles. Significantly increased enzymatic activity and release of proteasomes were observed in G6PD vs control RBCs. Similar variations were observed in stress protein biomarkers at the G6PD membrane. The proteasome profile (mainly the caspase-like activity) had significant correlations with the G6PD metabolome and quality markers of the RBC units. The storage-induced modifications in the proteasome activities were only partly restored in transfusion-mimicking conditions.
DISCUSSION
Storage conditions and G6PD deficiency affect (individually and in synergy) the abundance, distribution, activity, and release of RBC proteasomes. The partial irreversibility of these effects in transfusion-mimicking conditions demands further investigation of their clinical impact on transfusion outcomes.
Topics: Blood Preservation; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Humans; Oxidation-Reduction; Proteasome Endopeptidase Complex
PubMed: 33263521
DOI: 10.2450/2020.0179-20 -
Biomolecules Aug 2019The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells, it is required for the degradation of protein factors in multiple cellular... (Review)
Review
The 26S proteasome is the central element of proteostasis regulation in eukaryotic cells, it is required for the degradation of protein factors in multiple cellular pathways and it plays a fundamental role in cell stability. The main aspects of proteasome mediated protein degradation have been highly (but not totally) described during three decades of intense cellular, molecular, structural and chemical biology research and tool development. Contributions accumulated within this time lapse allow researchers today to go beyond classical partial views of the pathway, and start generating almost complete views of how the proteasome acts inside the cell. These views have been recently reinforced by cryo-electron microscopy and mechanistic works that provide from landscapes of proteasomal populations distributed in distinct intracellular contexts, to detailed shots of each step of the process of degradation of a given substrate, of the factors that regulate it, and precise measurements of the speed of degradation. Here, we present an updated digest of the most recent developments that significantly contribute in our understanding of how the 26S proteasome degrades hundreds of ubiquitinated substrates in multiple intracellular environments.
Topics: Animals; Cells; Humans; Proteasome Endopeptidase Complex; Protein Transport; Proteolysis; Ubiquitination
PubMed: 31443414
DOI: 10.3390/biom9090395 -
Current Opinion in Biotechnology Dec 2022Targeted protein degradation (TPD) is a broadly useful proteome editing tool for biological research and therapeutic development. TPD offers several advantages over... (Review)
Review
Targeted protein degradation (TPD) is a broadly useful proteome editing tool for biological research and therapeutic development. TPD offers several advantages over functional inhibition alone, including the ability to target previously undruggable proteins and the substantial and sustained knockout of protein activity. A variety of small molecule approaches hijack endogenous protein degradation machinery, but are limited to proteins with a cytosolic domain and suitable binding pocket. Recently, biologics-based methods have expanded the TPD toolbox by allowing access to extracellular and surface-exposed proteins and increasing target specificity. Here, we summarize recent advances in the use of biologics to deplete proteins through either the ubiquitin-proteasome system or the lysosomal degradation pathway, and discuss routes to their effective delivery as potential therapeutic interventions.
Topics: Proteolysis; Proteasome Endopeptidase Complex; Ubiquitin; Proteome
PubMed: 36179405
DOI: 10.1016/j.copbio.2022.102807 -
International Journal of Molecular... May 2020Protein folding is a substantively error prone process, especially when it occurs in the endoplasmic reticulum (ER). The highly exquisite machinery in the ER controls... (Review)
Review
Protein folding is a substantively error prone process, especially when it occurs in the endoplasmic reticulum (ER). The highly exquisite machinery in the ER controls secretory protein folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol; these misfolded proteins are then degraded by the ubiquitin-proteasome system termed as the ER-associated degradation (ERAD). The 26S proteasome is a multisubunit protease complex that recognizes and degrades ubiquitinated proteins in an ATP-dependent manner. The complex structure of the 26S proteasome requires exquisite regulation at the transcription, translation, and molecular assembly levels. Nuclear factor erythroid-derived 2-related factor 1 (Nrf1; NFE2L1), an ER-resident transcription factor, has recently been shown to be responsible for the coordinated expression of all the proteasome subunit genes upon proteasome impairment in mammalian cells. In this review, we summarize the current knowledge regarding the transcriptional regulation of the proteasome, as well as recent findings concerning the regulation of Nrf1 transcription activity in ER homeostasis and metabolic processes.
Topics: Animals; Endoplasmic Reticulum-Associated Degradation; Humans; Nuclear Respiratory Factor 1; Proteasome Endopeptidase Complex; Proteostasis
PubMed: 32456207
DOI: 10.3390/ijms21103683 -
Biomolecules Feb 2021PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates... (Review)
Review
PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28α, PA28β, and PA28γ, which assemble as heptameric hetero (PA28αβ) or homo (PA28γ) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28α and PA28β seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28αβ favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28γ seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28γ has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28αβ, most of these diverse biological functions of PA28γ seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αβ paralog.
Topics: Amino Acid Sequence; Animals; Atherosclerosis; Autoantigens; Humans; Isoenzymes; Models, Molecular; Neoplasms; Neurodegenerative Diseases; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Conformation; Protein Multimerization; Protein Subunits; Proteolysis; Proteostasis; Sequence Homology, Amino Acid; Ubiquitin
PubMed: 33562807
DOI: 10.3390/biom11020228 -
SLAS Discovery : Advancing Life... Aug 2020Aggresomes are subcellular perinuclear structures where misfolded proteins accumulate by retrograde transport on microtubules. Different methods are available to monitor...
Aggresomes are subcellular perinuclear structures where misfolded proteins accumulate by retrograde transport on microtubules. Different methods are available to monitor aggresome formation, but they are often laborious, time-consuming, and not quantitative. Proteostat is a red fluorescent molecular rotor dye, which becomes brightly fluorescent when it binds to protein aggregates. As this reagent was previously validated to detect aggresomes, we have miniaturized its use in 384-well plates and developed a method for high-throughput imaging and quantification of aggresomes. Two different image analysis methods, including one with machine learning, were evaluated. They lead to similar robust data to quantify cells having aggresome, with satisfactory Z' factor values and reproducible EC values for compounds known to induce aggresome formation, like proteasome inhibitors. We demonstrated the relevance of this phenotypic assay by screening a chemical library of 1280 compounds to find aggresome modulators. We obtained hits that present similarities in their structural and physicochemical properties. Interestingly, some of them were previously described to modulate autophagy, which could explain their effect on aggresome structures. In summary, we have optimized and validated the Proteostat detection reagent to easily measure aggresome formation in a miniaturized, automated, quantitative, and high-content assay. This assay can be used at low, middle, or high throughput to quantify changes in aggresome formation that could help in the understanding of chemical compound activity in pathologies such as protein misfolding disorders or cancer.
Topics: Autophagy; HeLa Cells; High-Throughput Screening Assays; Humans; Machine Learning; Microtubules; Molecular Imaging; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Aggregates
PubMed: 32449635
DOI: 10.1177/2472555220919708 -
Nature Aging May 2023Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective...
Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington's and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.
Topics: Animals; Humans; Proteasome Endopeptidase Complex; Longevity; Protein Aggregates; Caenorhabditis elegans; Cold Temperature; Trypsin
PubMed: 37118550
DOI: 10.1038/s43587-023-00383-4