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Biochimica Et Biophysica Acta Jan 2014In eukaryotic cells, the ubiquitin-proteasome-system (UPS) is responsible for the non-lysosomal degradation of proteins and plays a pivotal role in such vital processes... (Review)
Review
In eukaryotic cells, the ubiquitin-proteasome-system (UPS) is responsible for the non-lysosomal degradation of proteins and plays a pivotal role in such vital processes as protein homeostasis, antigen processing or cell proliferation. Therefore, it is an attractive drug target with various applications in cancer and immunosuppressive therapies. Being an evolutionary well conserved pathway, many pathogenic bacteria have developed small molecules, which modulate the activity of their hosts' UPS components. Such natural products are, due to their stepwise optimization over the millennia, highly potent in terms of their binding mechanisms, their bioavailability and selectivity. Generally, this makes bioactive natural products an ideal starting point for the development of novel drugs. Since four out of the ten best seller drugs are natural product derivatives, research in this field is still of unfathomable value for the pharmaceutical industry. The currently most prominent example for the successful exploitation of a natural compound in the UPS field is carfilzomib (Kyprolis®), which represents the second FDA approved drug targeting the proteasome after the admission of the blockbuster bortezomib (Velcade®) in 2003. On the other hand side of the spectrum, ONX 0914, which is derived from the same natural product as carfilzomib, has been shown to selectively inhibit the immune response related branch of the pathway. To date, there exists a huge potential of UPS inhibitors with regard to many diseases. Both approved drugs against the proteasome show severe side effects, adaptive resistances and limited applicability, thus the development of novel compounds with enhanced properties is a main objective of active research. In this review, we describe the techniques, which can be utilized for the discovery of novel natural inhibitors, which in particular block the 20S proteasomal activity. In addition, we will illustrate the successful implementation of a recently published methodology with the example of a highly potent but so far unexploited group of proteasome inhibitors, the syrbactins, and their biological functions. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Topics: Animals; Biological Products; Drug Evaluation, Preclinical; Humans; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Ubiquitination
PubMed: 23360979
DOI: 10.1016/j.bbamcr.2013.01.017 -
Cell May 2006Exosomes and proteasomes are macromolecular complexes that posttranscriptionally regulate gene expression by degrading mRNAs and proteins, respectively. Although the two... (Review)
Review
Exosomes and proteasomes are macromolecular complexes that posttranscriptionally regulate gene expression by degrading mRNAs and proteins, respectively. Although the two complexes act on different substrates and are composed of different subunits, they share a similar barrel-like architecture that appears to have evolved to restrict substrate access and prevent indiscriminate degradation.
Topics: Binding Sites; Exoribonucleases; Macromolecular Substances; Proteasome Endopeptidase Complex; Protein Conformation; Protein Subunits; RNA; Substrate Specificity
PubMed: 16713559
DOI: 10.1016/j.cell.2006.05.002 -
The Journal of Toxicological Sciences 2023Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding,...
Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.
Topics: Animals; Mice; Autophagy; Fibroblasts; Mercury; Methylmercury Compounds; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sequestosome-1 Protein; Ubiquitinated Proteins; Mercury Poisoning
PubMed: 37258240
DOI: 10.2131/jts.48.355 -
Biomolecules Mar 2022Severe COVID-19 disease leads to hypoxemia, inflammation and lymphopenia. Viral infection induces cellular stress and causes the activation of the innate immune...
Severe COVID-19 disease leads to hypoxemia, inflammation and lymphopenia. Viral infection induces cellular stress and causes the activation of the innate immune response. The ubiquitin-proteasome system (UPS) is highly implicated in viral immune response regulation. The main function of the proteasome is protein degradation in its active form, which recognises and binds to ubiquitylated proteins. Some proteasome subunits have been reported to be upregulated under hypoxic and hyperinflammatory conditions. Here, we conducted a prospective cohort study of COVID-19 patients ( = 44) and age-and sex-matched controls ( = 20). In this study, we suggested that hypoxia could induce the overexpression of certain genes encoding for subunits from the α and β core of the 20S proteasome and from regulatory particles (19S and 11S) in COVID-19 patients. Furthermore, the gene expression of proteasome subunits was associated with lymphocyte count reduction and positively correlated with inflammatory molecular and clinical markers. Given the importance of the proteasome in maintaining cellular homeostasis, including the regulation of the apoptotic and pyroptotic pathways, these results provide a potential link between COVID-19 complications and proteasome gene expression.
Topics: COVID-19; Humans; Hypoxia; Inflammation; Lymphopenia; Prospective Studies; Proteasome Endopeptidase Complex
PubMed: 35327634
DOI: 10.3390/biom12030442 -
Structure (London, England : 1993) Sep 2008The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S... (Review)
Review
The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, beta subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.
Topics: Animals; Catalytic Domain; Dimerization; Evolution, Molecular; Humans; Models, Biological; Models, Molecular; Molecular Chaperones; Multiprotein Complexes; Proteasome Endopeptidase Complex; Protein Binding; Protein Subunits
PubMed: 18786393
DOI: 10.1016/j.str.2008.07.001 -
Molecules (Basel, Switzerland) Jul 2021The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of...
The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of amide derivatives as non-covalent inhibitors of the β1i subunit with values in the low/submicromolar ranges have been identified. Here, we investigated the binding mechanism of the most potent and selective inhibitor, -benzyl-2-(2-oxopyridin-1(2H)-yl)propanamide (), to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400 ns of MD-binding analyses, followed by 200 ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together in a flipped orientation of a representative pose. Further, Binding Pose MetaDynamics (BPMD) studies were performed to evaluate the binding stability, comparing with four other inhibitors of the β1i subunit: -benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (), -cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propenamide (), -butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (), and ()-2-(2-oxopyridin-1(2H)-yl)-,4-diphenylbutanamide (). The obtained results in terms of free binding energy were consistent with the experimental values of inhibition, confirming as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events, and the information obtained could be exploited for the rational design of new and more active inhibitors.
Topics: Binding Sites; Dipeptides; Molecular Docking Simulation; Molecular Dynamics Simulation; Oligopeptides; Organosilicon Compounds; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding
PubMed: 34279386
DOI: 10.3390/molecules26134046 -
Polyubiquitin and ubiquitin-like signals share common recognition sites on proteasomal subunit Rpn1.The Journal of Biological Chemistry 2021Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL)...
Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.
Topics: Binding Sites; Cell Cycle Proteins; Cytoplasm; DNA-Binding Proteins; Humans; Magnetic Resonance Spectroscopy; Membrane Proteins; Polyubiquitin; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitins
PubMed: 33617881
DOI: 10.1016/j.jbc.2021.100450 -
Current Opinion in Structural Biology Apr 2020The 26S proteasome is the essential compartmental protease in eukaryotic cells required for the ubiquitin-dependent clearance of damaged polypeptides and obsolete... (Review)
Review
The 26S proteasome is the essential compartmental protease in eukaryotic cells required for the ubiquitin-dependent clearance of damaged polypeptides and obsolete regulatory proteins. Recently, a combination of high-resolution structural, biochemical, and biophysical studies has provided crucial new insights into the mechanisms of this fascinating molecular machine. A multitude of new cryo-electron microscopy structures provided snapshots of the proteasome during ATP-hydrolysis-driven substrate translocation, and detailed biochemical studies revealed the timing of individual degradation steps, elucidating the mechanisms for substrate selection and the commitment to degradation through conformational transitions. It was uncovered how ubiquitin removal from substrates is mechanically coupled to degradation, and cryo-electron tomography studies gave a glimpse of active proteasomes inside the cell, their subcellular localization, and interactions with protein aggregates. Here, we summarize these advances in our mechanistic understanding of the proteasome, with a particular focus on how its structural features and conformational transitions enable the multi-step degradation process.
Topics: Chemical Phenomena; Cryoelectron Microscopy; Humans; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Proteolysis; Structure-Activity Relationship; Substrate Specificity; Ubiquitination
PubMed: 31783300
DOI: 10.1016/j.sbi.2019.10.004 -
Biochimica Et Biophysica Acta Jan 2014The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto... (Review)
Review
The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by - at that time - an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Topics: Animals; Binding Sites; Humans; Models, Molecular; Polyubiquitin; Proteasome Endopeptidase Complex; Protein Binding; Protein Sorting Signals; Proteolysis; Substrate Specificity; Ubiquitinated Proteins
PubMed: 23872423
DOI: 10.1016/j.bbamcr.2013.07.007 -
TheScientificWorldJournal Aug 2010Heat shock proteins (HSPs) are chaperones that catalyze the proper folding of nascent proteins and the refolding of denatured proteins. The ubiquitin-proteasome system... (Review)
Review
Heat shock proteins (HSPs) are chaperones that catalyze the proper folding of nascent proteins and the refolding of denatured proteins. The ubiquitin-proteasome system is an error-checking system that directs improperly folded proteins for destruction. A coordinated interaction between the HSPs (renaturation) and the proteasome (degradation) must exist to assure protein quality control mechanisms. Although it still remains unknown how the decision of folding vs. degradation is taken, many pieces of evidence demonstrate that HSPs interact directly or indirectly with the proteasome, assuring quite selectively the proteasomal degradation of certain proteins under stress conditions. In this review, we will describe the different data that demonstrate a role for HSP90, HSP70, HSP27, and áB-crystallin in the partitioning of proteins to either one of these pathways, referred as protein triage.
Topics: Animals; Crystallins; HSP27 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Models, Biological; Proteasome Endopeptidase Complex; Protein Binding; Signal Transduction
PubMed: 20694452
DOI: 10.1100/tsw.2010.152