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Molecular Aspects of Medicine Aug 2016The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known... (Review)
Review
The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators.
Topics: Adaptation, Physiological; Aging; Animals; Catalysis; Disease Susceptibility; Humans; Metabolic Networks and Pathways; Oxidation-Reduction; Oxidative Stress; Proteasome Endopeptidase Complex; Protein Binding; Protein Subunits; Proteins; Proteolysis; Ubiquitin
PubMed: 27155164
DOI: 10.1016/j.mam.2016.05.001 -
Cells Aug 2021The selective targeting and disposal of solid protein aggregates are essential for cells to maintain protein homoeostasis. Autophagy receptors including p62, NBR1,... (Review)
Review
The selective targeting and disposal of solid protein aggregates are essential for cells to maintain protein homoeostasis. Autophagy receptors including p62, NBR1, Cue5/TOLLIP (CUET), and Tax1-binding protein 1 (TAX1BP1) proteins function in selective autophagy by targeting ubiquitinated aggregates through ubiquitin-binding domains. Here, we summarize previous beliefs and recent findings on selective receptors in aggregate autophagy. Since there are many reviews on selective autophagy receptors, we focus on their oligomerization, which enables receptors to function as pathway determinants and promotes phase separation.
Topics: Animals; Autophagosomes; Autophagy; Autophagy-Related Proteins; Humans; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Multimerization; Proteolysis; Proteostasis; Signal Transduction; Structure-Activity Relationship; Ubiquitination
PubMed: 34440758
DOI: 10.3390/cells10081989 -
International Journal of Molecular... Apr 2019Ubiquitin-like/ubiquitin-associated proteins (UbL-UbA) are a well-studied family of non-proteasomal ubiquitin receptors that are evolutionarily conserved across species.... (Review)
Review
Ubiquitin-like/ubiquitin-associated proteins (UbL-UbA) are a well-studied family of non-proteasomal ubiquitin receptors that are evolutionarily conserved across species. Members of this non-homogenous family facilitate and support proteasomal activity by promoting different effects on proteostasis but exhibit diverse extra-proteasomal activities. Dysfunctional UbL-UbA proteins render cells, particularly neurons, more susceptible to stressors or aging and may cause earlier neurodegeneration. In this review, we summarized the properties and functions of UbL-UbA family members identified to date, with an emphasis on new findings obtained using models showing a direct or indirect role in some neurodegenerative diseases.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy-Related Proteins; Carrier Proteins; Cell Cycle Proteins; DNA Repair Enzymes; DNA-Binding Proteins; Disease Models, Animal; Drosophila; Humans; Intracellular Signaling Peptides and Proteins; Neurodegenerative Diseases; Neurons; Proteasome Endopeptidase Complex; Proteostasis; Transcription Factors; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitins
PubMed: 30999567
DOI: 10.3390/ijms20081893 -
Cells Jan 2019The ubiquitin-proteasome system (UPS) and autophagy are the two major intracellular protein quality control (PQC) pathways that are responsible for cellular proteostasis... (Review)
Review
The ubiquitin-proteasome system (UPS) and autophagy are the two major intracellular protein quality control (PQC) pathways that are responsible for cellular proteostasis (homeostasis of the proteome) by ensuring the timely degradation of misfolded, damaged, and unwanted proteins. Ubiquitination serves as the degradation signal in both these systems, but substrates are precisely targeted to one or the other pathway. Determining how and when cells target specific proteins to these two alternative PQC pathways and control the crosstalk between them are topics of considerable interest. The ubiquitin (Ub) recognition code based on the type of Ub-linked chains on substrate proteins was believed to play a pivotal role in this process, but an increasing body of evidence indicates that the PQC pathway choice is also made based on other criteria. These include the oligomeric state of the Ub-binding protein shuttles, their conformation, protein modifications, and the presence of motifs that interact with ATG8/LC3/GABARAP (autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3/GABA type A receptor-associated protein) protein family members. In this review, we summarize the current knowledge regarding the Ub recognition code that is bound by Ub-binding proteasomal and autophagic receptors. We also discuss how cells can modify substrate fate by modulating the structure, conformation, and physical properties of these receptors to affect their shuttling between both degradation pathways.
Topics: Animals; Autophagy; Humans; Plants; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin; Ubiquitin-Protein Ligase Complexes; Ubiquitinated Proteins; Ubiquitination; Yeasts
PubMed: 30634694
DOI: 10.3390/cells8010040 -
The FEBS Journal Dec 2016Plasmodium falciparum is the parasite responsible for the most severe form of malaria. Its increasing resistance to existing antimalarials represents a major threat to... (Review)
Review
Plasmodium falciparum is the parasite responsible for the most severe form of malaria. Its increasing resistance to existing antimalarials represents a major threat to human health and urges the development of new therapeutic strategies to fight malaria. The proteasome is a protease complex essential in all eukaryotes. Accordingly, inhibition of the Plasmodium 20S proteasome is highly toxic for the parasite at all of its infective and developmental stages. Proteasome inhibitors have antimalarial potential both as curative and transmission blocking agents, but in order to have therapeutic application, they must specifically target the Plasmodium proteasome and not its human counterpart. X-ray crystallography has been widely used to determine structures of yeast and mammalian 20S proteasomes with ligands. However, crystallisation of the Plasmodium proteasome is challenging, as only small quantities of the complex can be directly purified from the parasite. Furthermore, most X-ray structures of proteasome-inhibitor complexes require soaking of crystals with high concentrations of ligand, thus preventing analysis of inhibitor subunit specificity. Instead we chose to determine the Plasmodium falciparum 20S proteasome structure, in the presence of a new rationally designed parasite-specific inhibitor, by high-resolution electron cryo-microscopy and single particle analysis. The resulting map, at a resolution of about 3.6 Å, allows a direct molecular analysis of inhibitor/enzyme interactions. Here we present an overview of this structure, and how it provides valuable information that can be used to assist in the design of improved proteasome inhibitors with the potential to be developed as next-generation antimalarial drugs.
Topics: Cryoelectron Microscopy; Host-Parasite Interactions; Humans; Malaria, Falciparum; Models, Molecular; Plasmodium falciparum; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Conformation
PubMed: 27286897
DOI: 10.1111/febs.13780 -
PLoS Genetics May 2023Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most...
Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most protein degradation occurs through the stepwise actions of two functionally distinct entities, the ubiquitin system and the proteasome. Ubiquitin system enzymes attach ubiquitin to cellular proteins, targeting them for degradation. The proteasome then selectively binds and degrades ubiquitinated substrate proteins. Genetic variation in ubiquitin system genes creates heritable differences in the degradation of their substrates. However, the challenges of measuring the degradative activity of the proteasome independently of the ubiquitin system in large samples have limited our understanding of genetic influences on the proteasome. Here, using the yeast Saccharomyces cerevisiae, we built and characterized reporters that provide high-throughput, ubiquitin system-independent measurements of proteasome activity. Using single-cell measurements of proteasome activity from millions of genetically diverse yeast cells, we mapped 15 loci across the genome that influence proteasomal protein degradation. Twelve of these 15 loci exerted specific effects on the degradation of two distinct proteasome substrates, revealing a high degree of substrate-specificity in the genetics of proteasome activity. Using CRISPR-Cas9-based allelic engineering, we resolved a locus to a causal variant in the promoter of RPT6, a gene that encodes a subunit of the proteasome's 19S regulatory particle. The variant increases RPT6 expression, which we show results in increased proteasome activity. Our results reveal the complex genetic architecture of proteasome activity and suggest that genetic influences on the proteasome may be an important source of variation in the many cellular and organismal traits shaped by protein degradation.
Topics: Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Proteolysis; Ubiquitin; Saccharomyces cerevisiae Proteins; Genetic Variation
PubMed: 37126494
DOI: 10.1371/journal.pgen.1010734 -
Traffic (Copenhagen, Denmark) May 2022Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular...
Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p-proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc-proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune-subunits) and intermediate (containing some immune-subunits) forms of 20S are observed. Moreover, we show that galectin-3, which displays a highly disordered N-terminal region, is efficiently cleaved by purified cc-proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin-3 is unable to induce red blood cells agglutination when preincubated with cc-proteasome. These results highlight potential novel physio- and pathologic functions for the extracellular proteasome.
Topics: Agglutination; Cytoplasm; Galectin 3; Humans; Proteasome Endopeptidase Complex; Proteolysis
PubMed: 35466519
DOI: 10.1111/tra.12840 -
Biomolecules Jan 2021Four decades of proteasome research have yielded extensive information on ubiquitin-dependent proteolysis. The archetype of proteasomes is a 20S barrel-shaped complex... (Review)
Review
Four decades of proteasome research have yielded extensive information on ubiquitin-dependent proteolysis. The archetype of proteasomes is a 20S barrel-shaped complex that does not rely on ubiquitin as a degradation signal but can degrade substrates with a considerable unstructured stretch. Since roughly half of all proteasomes in most eukaryotic cells are free 20S complexes, ubiquitin-independent protein degradation may coexist with ubiquitin-dependent degradation by the highly regulated 26S proteasome. This article reviews recent advances in our understanding of the biochemical and structural features that underlie the proteolytic mechanism of 20S proteasomes. The two outer α-rings of 20S proteasomes provide a number of potential docking sites for loosely folded polypeptides. The binding of a substrate can induce asymmetric conformational changes, trigger gate opening, and initiate its own degradation through a protease-driven translocation mechanism. Consequently, the substrate translocates through two additional narrow apertures augmented by the β-catalytic active sites. The overall pulling force through the two annuli results in a protease-like unfolding of the substrate and subsequent proteolysis in the catalytic chamber. Although both proteasomes contain identical β-catalytic active sites, the differential translocation mechanisms yield distinct peptide products. Nonoverlapping substrate repertoires and product outcomes rationalize cohabitation of both proteasome complexes in cells.
Topics: Catalysis; Catalytic Domain; Cytoplasm; Humans; Intrinsically Disordered Proteins; Microscopy, Atomic Force; Oxidative Stress; Peptides; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Protein Domains; Protein Processing, Post-Translational; Proteolysis; Proteome; Substrate Specificity; Ubiquitin; Ubiquitination
PubMed: 33498876
DOI: 10.3390/biom11020148 -
The Journal of Biological Chemistry Dec 2023Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy...
Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.
Topics: Humans; Proteasome Endopeptidase Complex; Proteins; Ubiquitin-Protein Ligases; Proteolysis; Ubiquitination; Ubiquitin
PubMed: 37866632
DOI: 10.1016/j.jbc.2023.105381 -
ELife Jan 2022The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual...
The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits (Erzberger and Berger, 2006; Puchades et al., 2020). How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.
Topics: Adenosine Triphosphatases; Energy Metabolism; Kinetics; Models, Molecular; Peptides; Proteasome Endopeptidase Complex; Protein Conformation; Saccharomyces cerevisiae; Substrate Specificity; Translocation, Genetic
PubMed: 35050852
DOI: 10.7554/eLife.71911