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Annual Review of Pharmacology and... Jan 2020Proteasomes are large, multicatalytic protein complexes that cleave cellular proteins into peptides. There are many distinct forms of proteasomes that differ in... (Review)
Review
Proteasomes are large, multicatalytic protein complexes that cleave cellular proteins into peptides. There are many distinct forms of proteasomes that differ in catalytically active subunits, regulatory subunits, and associated proteins. Proteasome inhibitors are an important class of drugs for the treatment of multiple myeloma and mantle cell lymphoma, and they are being investigated for other diseases. Bortezomib (Velcade) was the first proteasome inhibitor to be approved by the US Food and Drug Administration. Carfilzomib (Kyprolis) and ixazomib (Ninlaro) have recently been approved, and more drugs are in development. While the primary mechanism of action is inhibition of the proteasome, the downstream events that lead to selective cell death are not entirely clear. Proteasome inhibitors have been found to affect protein turnover but at concentrations that are much higher than those achieved clinically, raising the possibility that some of the effects of proteasome inhibitors are mediated by other mechanisms.
Topics: Animals; Antineoplastic Agents; Drug Development; Humans; Lymphoma, Mantle-Cell; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors
PubMed: 31479618
DOI: 10.1146/annurev-pharmtox-010919-023603 -
Biomolecules Jan 2021The ubiquitin-proteasome system (UPS) is the major intracellular and non-lysosomal protein degradation system. Thanks to its unique capacity of eliminating old, damaged,... (Review)
Review
The ubiquitin-proteasome system (UPS) is the major intracellular and non-lysosomal protein degradation system. Thanks to its unique capacity of eliminating old, damaged, misfolded, and/or regulatory proteins in a highly specific manner, the UPS is virtually involved in almost all aspects of eukaryotic life. The critical importance of the UPS is particularly visible in immune cells which undergo a rapid and profound functional remodelling upon pathogen recognition. Innate and/or adaptive immune activation is indeed characterized by a number of substantial changes impacting various cellular processes including protein homeostasis, signal transduction, cell proliferation, and antigen processing which are all tightly regulated by the UPS. In this review, we summarize and discuss recent progress in our understanding of the molecular mechanisms by which the UPS contributes to the generation of an adequate immune response. In this regard, we also discuss the consequences of UPS dysfunction and its role in the pathogenesis of recently described immune disorders including cancer and auto-inflammatory diseases.
Topics: Animals; Antigen Presentation; Humans; Leukocytes; Proteasome Endopeptidase Complex; Proteostasis; Signal Transduction; Ubiquitin
PubMed: 33466553
DOI: 10.3390/biom11010060 -
Molecular Cancer Apr 2022Proteolysis-targeting chimeras (PROTACs) are engineered techniques for targeted protein degradation. A bifunctional PROTAC molecule with two covalently-linked ligands... (Review)
Review
Proteolysis-targeting chimeras (PROTACs) are engineered techniques for targeted protein degradation. A bifunctional PROTAC molecule with two covalently-linked ligands recruits target protein and E3 ubiquitin ligase together to trigger proteasomal degradation of target protein by the ubiquitin-proteasome system. PROTAC has emerged as a promising approach for targeted therapy in various diseases, particularly in cancers. In this review, we introduce the principle and development of PROTAC technology, as well as the advantages of PROTACs over traditional anti-cancer therapies. Moreover, we summarize the application of PROTACs in targeting critical oncoproteins, provide the guidelines for the molecular design of PROTACs and discuss the challenges in the targeted degradation by PROTACs.
Topics: Humans; Ligands; Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin-Protein Ligases
PubMed: 35410300
DOI: 10.1186/s12943-021-01434-3 -
Nature Communications May 2022The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely...
The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Here, we compare the contributions of the proteasome and DUBs on the global ubiquitinome, using UbiSite technology, inhibitors and mass spectrometry. We uncover large dynamic ubiquitin signalling networks with substrates and sites preferentially regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitination. DUBs regulate substrates via at least 40,000 unique sites. Regulated networks of ubiquitin substrates are involved in autophagy, apoptosis, genome integrity, telomere integrity, cell cycle progression, mitochondrial function, vesicle transport, signal transduction, transcription, pre-mRNA splicing and many other cellular processes. Moreover, we show that ubiquitin conjugated to SUMO2/3 forms a strong proteasomal degradation signal. Interestingly, PARP1 is hyper-ubiquitinated in response to DUB inhibition, which increases its enzymatic activity. Our study uncovers key regulatory roles of DUBs and provides a resource of endogenous ubiquitination sites to aid the analysis of substrate specific ubiquitin signalling.
Topics: Cell Division; Deubiquitinating Enzymes; Proteasome Endopeptidase Complex; Ubiquitin; Ubiquitination
PubMed: 35585066
DOI: 10.1038/s41467-022-30376-7 -
Nature Feb 2020The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related...
The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
Topics: Cell Line; Cell Nucleus; DNA Repair Enzymes; DNA-Binding Proteins; Humans; Osmotic Pressure; Polyubiquitin; Proteasome Endopeptidase Complex; Proteolysis; Proteostasis; Ribosomal Proteins; Stress, Physiological; Ubiquitin-Protein Ligases; Ubiquitination; Valosin Containing Protein
PubMed: 32025036
DOI: 10.1038/s41586-020-1982-9 -
Molecular Cell Jun 2023Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent...
Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.
Topics: Humans; Ubiquitin; Proteolysis; Proteasome Endopeptidase Complex; Proteins; Ubiquitination; Cell Cycle Proteins
PubMed: 37201526
DOI: 10.1016/j.molcel.2023.04.023 -
Nature Nov 2021Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial...
Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
Topics: Active Transport, Cell Nucleus; CRISPR-Cas Systems; Cell Line, Tumor; Cell Nucleus; DNA-Binding Proteins; Female; Genes, myc; Humans; Male; Mitosis; Nuclear Proteins; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Transcription Factors
PubMed: 34711951
DOI: 10.1038/s41586-021-04035-8 -
Journal of the American Chemical Society Jun 2023Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either...
Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either ubiquitin-proteasome or lysosomal systems, thus are powerless against target proteins in membrane organelles lacking proteasomes and lysosomes, such as mitochondria. Here, we developed a mitochondrial protease targeting chimera (MtPTAC) to address this issue. MtPTAC is a bifunctional small molecule that can bind to mitochondrial caseinolytic protease P (ClpP) at one end and target protein at the other. Mechanistically, MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing target proteins into proximity with ClpP. Taking mitochondrial RNA polymerase (POLRMT) as a model protein, we have demonstrated the powerful proteolytic ability and antitumor application prospects of MtPTAC, both and . This is the first modularly designed TPD that can specifically hydrolyze target proteins inside mitochondria.
Topics: Proteolysis; Mitochondria; Proteins; Proteasome Endopeptidase Complex; Ubiquitin; Endopeptidases
PubMed: 37276358
DOI: 10.1021/jacs.3c03756 -
Science (New York, N.Y.) May 2023The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy,...
The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19) and catalytic (20) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19 particles near synapses. The free neuronal 19 particles bind and deubiquitylate lysine 63-ubiquitin (Lys-ub), a non-proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys-ub interactors. Inhibition of the 19 deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.
Topics: Animals; Rats; Neurons; Proteasome Endopeptidase Complex; Proteolysis; Synapses; Ubiquitin; Lysine; Synaptic Transmission
PubMed: 37228199
DOI: 10.1126/science.adf2018 -
Nature Chemical Biology Jan 2023Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of...
Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.
Topics: Nuclear Proteins; Cryoelectron Microscopy; Transcription Factors; Proteasome Endopeptidase Complex; Proteolysis; Ligases; Ubiquitin-Protein Ligases
PubMed: 36577875
DOI: 10.1038/s41589-022-01218-w