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Chemical Society Reviews Jun 2022Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of one ligand that binds to a protein of interest (POI) and another that can recruit... (Review)
Review
Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of one ligand that binds to a protein of interest (POI) and another that can recruit an E3 ubiquitin ligase. The chemically-induced proximity between the POI and E3 ligase results in ubiquitination and subsequent degradation of the POI by the ubiquitin-proteasome system (UPS). The event-driven mechanism of action (MOA) of PROTACs offers several advantages compared to traditional occupancy-driven small molecule inhibitors, such as a catalytic nature, reduced dosing and dosing frequency, a more potent and longer-lasting effect, an added layer of selectivity to reduce potential toxicity, efficacy in the face of drug-resistance mechanisms, targeting nonenzymatic functions, and expanded target space. Here, we highlight important milestones and briefly discuss lessons learned about targeted protein degradation (TPD) in recent years and conjecture on the efforts still needed to expand the toolbox for PROTAC discovery to ultimately provide promising therapeutics.
Topics: Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35671157
DOI: 10.1039/d2cs00193d -
Science (New York, N.Y.) Nov 2019To achieve homeostasis, cells evolved dynamic and self-regulating quality control processes to adapt to new environmental conditions and to prevent prolonged damage. We... (Review)
Review
To achieve homeostasis, cells evolved dynamic and self-regulating quality control processes to adapt to new environmental conditions and to prevent prolonged damage. We discuss the importance of two major quality control systems responsible for degradation of proteins and organelles in eukaryotic cells: the ubiquitin-proteasome system (UPS) and autophagy. The UPS and autophagy form an interconnected quality control network where decision-making is self-organized on the basis of biophysical parameters (binding affinities, local concentrations, and avidity) and compartmentalization (through membranes, liquid-liquid phase separation, or the formation of aggregates). We highlight cellular quality control factors that delineate their differential deployment toward macromolecular complexes, liquid-liquid phase-separated subcellular structures, or membrane-bound organelles. Finally, we emphasize the need for continuous promotion of quantitative and mechanistic research into the roles of the UPS and autophagy in human pathophysiology.
Topics: Animals; Autophagy; Humans; Macroautophagy; Organelles; Proteasome Endopeptidase Complex; Proteolysis; Ubiquitin
PubMed: 31727826
DOI: 10.1126/science.aax3769 -
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 -
Nature Medicine Feb 2018The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this...
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Topics: Animals; Cell Line, Tumor; DNA Damage; DNA Repair; Humans; Imides; Mice; Neoplasms; Nucleosides; Proteasome Endopeptidase Complex; Protein Binding; Pyrazoles; Pyrimidines; Small Molecule Libraries; Sulfides; Sulfonamides; Ubiquitin; Ubiquitin-Activating Enzymes; Xenograft Model Antitumor Assays
PubMed: 29334375
DOI: 10.1038/nm.4474 -
Nature Reviews. Molecular Cell Biology Jun 2019The deubiquitylating enzymes (DUBs, also known as deubiquitylases or deubiquitinases) maintain the dynamic state of the cellular ubiquitome by releasing conjugated... (Review)
Review
The deubiquitylating enzymes (DUBs, also known as deubiquitylases or deubiquitinases) maintain the dynamic state of the cellular ubiquitome by releasing conjugated ubiquitin from proteins. In light of the many cellular functions of ubiquitin, DUBs occupy key roles in almost all aspects of cell behaviour. Many DUBs show selectivity for particular ubiquitin linkage types or positions within ubiquitin chains. Others show chain-type promiscuity but can select a distinct palette of protein substrates via specific protein-protein interactions established through binding modules outside of the catalytic domain. The ubiquitin chain cleavage mode or chain linkage specificity has been related directly to biological functions. Examples include regulation of protein degradation and ubiquitin recycling by the proteasome, DNA repair pathways and innate immune signalling. DUB cleavage specificity is also being harnessed for analysis of ubiquitin chain architecture that is assembled on specific proteins. The recent development of highly specific DUB inhibitors heralds their emergence as a new class of therapeutic targets for numerous diseases.
Topics: Animals; Deubiquitinating Enzymes; Humans; Proteasome Endopeptidase Complex; Proteolysis; Signal Transduction; Substrate Specificity; Ubiquitin; Ubiquitination
PubMed: 30733604
DOI: 10.1038/s41580-019-0099-1 -
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 -
Cellular and Molecular Life Sciences :... Dec 2014In eukaryotic cells, proteasomes are highly conserved protease complexes and eliminate unwanted proteins which are marked by poly-ubiquitin chains for degradation. The... (Review)
Review
In eukaryotic cells, proteasomes are highly conserved protease complexes and eliminate unwanted proteins which are marked by poly-ubiquitin chains for degradation. The 26S proteasome consists of the proteolytic core particle, the 20S proteasome, and the 19S regulatory particle, which are composed of 14 and 19 different subunits, respectively. Proteasomes are the second-most abundant protein complexes and are continuously assembled from inactive precursor complexes in proliferating cells. The modular concept of proteasome assembly was recognized in prokaryotic ancestors and applies to eukaryotic successors. The efficiency and fidelity of eukaryotic proteasome assembly is achieved by several proteasome-dedicated chaperones that initiate subunit incorporation and control the quality of proteasome assemblies by transiently interacting with proteasome precursors. It is important to understand the mechanism of proteasome assembly as the proteasome has key functions in the turnover of short-lived proteins regulating diverse biological processes.
Topics: Animals; Humans; Models, Biological; Models, Molecular; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Protein Subunits
PubMed: 25107634
DOI: 10.1007/s00018-014-1699-8 -
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 -
Chemical Society Reviews Oct 2022The von Hippel-Lindau (VHL) Cullin RING E3 ligase is an essential enzyme in the ubiquitin-proteasome system that recruits substrates such as the hypoxia inducible factor... (Review)
Review
The von Hippel-Lindau (VHL) Cullin RING E3 ligase is an essential enzyme in the ubiquitin-proteasome system that recruits substrates such as the hypoxia inducible factor for ubiquitination and subsequent proteasomal degradation. The ubiquitin-proteasome pathway can be hijacked toward non-native neo-substrate proteins using proteolysis targeting chimeras (PROTACs), bifunctional molecules designed to simultaneously bind to an E3 ligase and a target protein to induce target ubiquitination and degradation. The availability of high-quality small-molecule ligands with good binding affinity for E3 ligases is fundamental for PROTAC development. Lack of good E3 ligase ligands as starting points to develop PROTAC degraders was initially a stumbling block to the development of the field. Herein, the journey towards the design of small-molecule ligands binding to VHL is presented. We cover the structure-based design of VHL ligands, their application as inhibitors in their own right, and their implementation into rationally designed, potent PROTAC degraders of various target proteins. We highlight the key findings and learnings that have provided strong foundations for the remarkable development of targeted protein degradation, and that offer a blueprint for designing new ligands for E3 ligases beyond VHL.
Topics: Cullin Proteins; Ligands; Proteasome Endopeptidase Complex; Ubiquitin; Von Hippel-Lindau Tumor Suppressor Protein
PubMed: 35983982
DOI: 10.1039/d2cs00387b -
Small (Weinheim An Der Bergstrasse,... Mar 2023Understanding the direct interaction of nanostructures per se with biological systems is important for biomedical applications. However, whether nanostructures regulate...
Understanding the direct interaction of nanostructures per se with biological systems is important for biomedical applications. However, whether nanostructures regulate biological systems by targeting specific cellular proteins remains largely unknown. In the present work, self-assembling nanomicelles are constructed using small-molecule oleanolic acid (OA) as a molecular template. Unexpectedly, without modifications by functional ligands, OA nanomicelles significantly activate cellular proteasome function by directly binding to 20S proteasome subunit alpha 6 (PSMA6). Mechanism study reveals that OA nanomicelles interact with PSMA6 to dynamically modulate its N-terminal domain conformation change, thereby controlling the entry of proteins into 20S proteasome. Subsequently, OA nanomicelles accelerate the degradation of several crucial proteins, thus potently driving cancer cell pyroptosis. For translational medicine, OA nanomicelles exhibit a significant anticancer potential in tumor-bearing mouse models and stimulate immune cell infiltration. Collectively, this proof-of-concept study advances the mechanical understanding of nanostructure-guided biological effects via their inherent capacity to activate proteasome.
Topics: Animals; Mice; Proteasome Endopeptidase Complex; Pyroptosis; Cytoplasm; Micelles; Nanostructures; Neoplasms
PubMed: 36549896
DOI: 10.1002/smll.202205531