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Journal of Biochemistry Mar 2022Most regulated proteolysis in cells is conducted by the ubiquitin-proteasome system (UPS), in which proteins to be eliminated are selected through multiple steps to... (Review)
Review
Most regulated proteolysis in cells is conducted by the ubiquitin-proteasome system (UPS), in which proteins to be eliminated are selected through multiple steps to achieve high specificity. The large protease complex proteasome binds to ubiquitin molecules that are attached to the substrate and further interacts with a disordered region in the target to initiate unfolding for degradation. Recent studies have expanded our view of the complexity of ubiquitination as well as the details of substrate engagement by the proteasome and at the same time have suggested the characteristics of substrates that are susceptible to proteasomal degradation. Here, I review some destabilizing elements of proteasome substrates with particular attention to ubiquitination, initiation region and stability against unfolding and discuss their interplay to determine the substrate stability. A spatial perspective is important to understand the mechanism of action of proteasomal degradation, which may be critical for drug development targeting the UPS including targeted protein degradation.
Topics: Proteasome Endopeptidase Complex; Proteolysis; Substrate Specificity; Ubiquitin; Ubiquitination
PubMed: 34967398
DOI: 10.1093/jb/mvab157 -
Methods in Molecular Biology (Clifton,... 2015Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved...
Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.
Topics: Animals; Caspases; Cell Extracts; Humans; Luciferases; Luminescent Measurements; Proteasome Endopeptidase Complex
PubMed: 25308265
DOI: 10.1007/978-1-4939-1661-0_9 -
Biomolecules Jan 2022The proteasome is responsible for selective degradation of most cellular proteins. Abundantly present in the cell, proteasomes not only diffuse in the cytoplasm and the... (Review)
Review
The proteasome is responsible for selective degradation of most cellular proteins. Abundantly present in the cell, proteasomes not only diffuse in the cytoplasm and the nucleus but also associate with the chromatin, cytoskeleton, various membranes and membraneless organelles/condensates. How and why the proteasome gets to these specific subcellular compartments remains poorly understood, although increasing evidence supports the hypothesis that intracellular localization may have profound impacts on the activity, substrate accessibility and stability/integrity of the proteasome. In this short review, I summarize recent advances on the functions, regulations and targeting mechanisms of proteasomes, especially those localized to the nuclear condensates and membrane structures of the cell, and I discuss the biological significance thereof in mediating compartmentalized protein degradation.
Topics: Cell Nucleus; Cytoplasm; Proteasome Endopeptidase Complex; Proteins; Proteolysis
PubMed: 35204730
DOI: 10.3390/biom12020229 -
Biomeditsinskaia Khimiia Mar 2018Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular... (Review)
Review
Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: the ATP-dependent addition of (typically four sequential) residues of the low-molecular ubiquitin protein, involving the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme and ubiquitin ligase. The cytoplasm and nucleoplasm proteins labeled in this way are then digested in 26S proteasomes. However, in recent years it has become increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent protein degradation in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate further increase. Since 26S of proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles and whole cells, has the most serious consequences for the whole organism.
Topics: Animals; Humans; Proteasome Endopeptidase Complex; Proteins; Proteolysis; Ubiquitin; Ubiquitination
PubMed: 29723144
DOI: 10.18097/PBMC20186402134 -
International Journal of Molecular... Jul 2020The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of... (Review)
Review
The 26S proteasome, a master player in proteolysis, is the most complex and meticulously contextured protease in eukaryotic cells. While capable of hosting thousands of discrete substrates due to the selective recognition of ubiquitin tags, this protease complex is also dynamically checked through diverse regulatory mechanisms. The proteasome's versatility ensures precise control over active proteolysis, yet prevents runaway or futile degradation of many essential cellular proteins. Among the multi-layered processes regulating the proteasome's proteolysis, deubiquitination reactions are prominent because they not only recycle ubiquitins, but also impose a critical checkpoint for substrate degradation on the proteasome. Of note, three distinct classes of deubiquitinating enzymes-USP14, RPN11, and UCH37-are associated with the 19S subunits of the human proteasome. Recent biochemical and structural studies suggest that these enzymes exert dynamic influence over proteasome output with limited redundancy, and at times act in opposition. Such distinct activities occur spatially on the proteasome, temporally through substrate processing, and differentially for ubiquitin topology. Therefore, deubiquitinating enzymes on the proteasome may fine-tune the degradation depending on various cellular contexts and for dynamic proteolysis outcomes. Given that the proteasome is among the most important drug targets, the biology of proteasome-associated deubiquitination should be further elucidated for its potential targeting in human diseases.
Topics: Humans; Proteasome Endopeptidase Complex; Trans-Activators; Ubiquitin; Ubiquitin Thiolesterase
PubMed: 32726943
DOI: 10.3390/ijms21155312 -
Sub-cellular Biochemistry 2021The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades... (Review)
Review
The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal "processor" for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.
Topics: Humans; Proteasome Endopeptidase Complex; Protein Conformation; Ubiquitin; Ubiquitination
PubMed: 33252727
DOI: 10.1007/978-3-030-58971-4_1 -
Biomolecules Aug 2014The 26S proteasome is an integral element of the ubiquitin-proteasome system(UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells.It... (Review)
Review
The 26S proteasome is an integral element of the ubiquitin-proteasome system(UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells.It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates.The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases,shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship.Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS).
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Proteasome Endopeptidase Complex; Protein Conformation; Proteolysis
PubMed: 25102382
DOI: 10.3390/biom4030774 -
IUBMB Life Jan 2022The tight regulation of proteostasis is essential for physiological cellular function. Mammalian cells possess a network of mechanisms that ensure proteome integrity... (Review)
Review
The tight regulation of proteostasis is essential for physiological cellular function. Mammalian cells possess a network of mechanisms that ensure proteome integrity under normal or stress conditions. The proteasome, being the major cellular proteolytic machinery, is central to proteostasis maintenance in response to distinct intracellular and extracellular conditions. The proteasomes are multisubunit protease complexes that selectively catalyze the degradation of short-lived regulatory proteins and damaged peptides. Different forms of the proteasome complexes comprising of different subunits and attached regulators directly affect the substrate selectivity and degradation. Thus, the proteasome participates in the turnover of a multitude of factors that control key processes that affect the cellular state, such as adaptation to environmental cues, growth, development, metabolism, signaling, senescence, pluripotency, differentiation, and immunity. Aberrations on its function are related to normal processes like aging and pathological conditions such as neurodegeneration and cancer. The past few years of research have highlighted that proteasome abundance, activity, assembly, and localization are subject to a dynamic transcriptional control that secures the continuous adaptation of the proteasome to internal or external stimuli. This review focuses on the factors and signaling pathways that are involved in the regulation of the mammalian proteasome at the transcriptional level. A comprehensive understanding of proteasome regulation has critical implications on disease prevention and treatment.
Topics: Aging; Animals; Gene Regulatory Networks; Mammals; Proteasome Endopeptidase Complex; Transcription Factors
PubMed: 34958522
DOI: 10.1002/iub.2586 -
The FEBS Journal May 2017Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes,... (Review)
Review
Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents.
Topics: Animals; Anti-Infective Agents; Humans; Mycobacterium; Plasmodium; Proteasome Endopeptidase Complex; Schistosoma
PubMed: 28122162
DOI: 10.1111/febs.14029 -
Current Protocols Apr 2023In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently...
In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purification schemes for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae. A gel filtration step can be added to further enhance purity. We also describe assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Purification of active 26S proteasomes Support Protocol 1: Growth of yeast strains and production of yeast cell powder Support Protocol 2: Regeneration of anti-flag M2 affinity gel Basic Protocol 2: Purification of the 19S regulatory particle (RP) Basic Protocol 3: Purification of active 20S CP Basic Protocol 4: In-gel peptidase activity assay for 20S CP and 26S proteasomes Basic Protocol 5: In-solution peptidase activity assay for 20S and 26S proteasomes Basic Protocol 6: Measuring degradation of polyubiquitinated SIC1 Basic Protocol 7: Gel filtration of purified proteasomes and subcomplexes.
Topics: Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Ubiquitin; Cytoplasm; Yeast, Dried
PubMed: 37026813
DOI: 10.1002/cpz1.717