Jin, L. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Grice, G. The proteasome distinguishes between heterotypic and homotypic lysinelinked polyubiquitin chains. Cell Rep. Meyer, H. Enhanced protein degradation by branched ubiquitin chains.
Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control. Cell , — Deveraux, Q.
A 26S protease subunit that binds ubiquitin conjugates. Husnjak, K. Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature , — Schreiner, P. Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Shi, Y. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science , aad Lam, Y. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal.
Paraskevopoulos, K. Dss1 is a 26S proteasome ubiquitin receptor. Cell 56 , — Fu, H. Structure and functional analysis of the 26S proteasome subunits from plants. Young, P. Characterization of two polyubiquitin binding sites in the 26 S protease subunit 5a.
Elsasser, S. Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Identification of ubiquitin-like protein-binding subunits of the 26S proteasome. Rosenzweig, R. Rpn1 and Rpn2 coordinate ubiquitin processing factors at proteasome. Chen, X et al. Structure 24 , — Bertolaet, B. Wilkinson, C. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Chen, L. Ubiquitin-associated UBA domains in Rad23 bind ubiquitin and promote inhibition of multi-ubiquitin chain assembly.
EMBO Rep. Rao, H. Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad Raasi, S. Verma, R.
Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell , 99— Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. Itakura, E. Ubiquilins chaperone and triage mitochondrial membrane proteins for degradation.
Cell 63 , 21—33 Hjerpe, R. UBQLN2 mediates autophagy-independent protein aggregate clearance by the proteasome. Sakata, E. The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle. Cell 42 , — Bashore, C. Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome.
Nager, A. Leggett, D. Multiple associated proteins regulate proteasome structure and function. Cell 10 , — Ziv, I. A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis. Cell Proteomics 10 , M Wickliffe, K. Klinked ubiquitin chains as novel regulators of cell division. Trends Cell Biol. Iwai, K. Linear polyubiquitination: a new regulator of NF-kappaB activation. Specific modification of aged proteasomes revealed by Tag-exchangeable knock-in mice.
Google Scholar. Stack, J. A ubiquitin-based tagging system for controlled modulation of protein stability. Koodathingal, P. ATP-dependent proteases differ substantially in their ability to unfold globular proteins. Kraut, D. Sequence- and species-dependence of proteasomal processivity. ACS Chem. Lu, Y. Substrate degradation by the proteasome: a single-molecule kinetic analysis. Science , Braten O. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.
USA , E Zhang, N. Structure of the S5a:Klinked diubiquitin complex and its interactions with Rpn Cell 35 , — Lee, B. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Meddling with fate: the proteasomal deubiquitinating enzymes.
Hamazaki, J. A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. Qiu, X. Yao, T. Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1.
Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Ding, Z. Cell Res. Wehmer, M. Structural insights into the functional cycle of the ATPase module of the 26S proteasome. Natl Acad. USA , — Huang, X. An atomic structure of the human 26S proteasome. Schweitzer, A. Structure of the human 26S proteasome at a resolution of 3.
Luan, B. Structure of an endogenous yeast 26S proteasome reveals two major conformational states. Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy. Walters, K. Petroski, M. Context of multiubiquitin chain attachment influences the rate of Sic1 degradation.
Cell 11 , — Pickart, C. Controlled synthesis of polyubiquitin chains. Methods Enzymol. Cannon, J. Top-down nm ultraviolet photodissociation mass spectrometry for simultaneous determination of polyubiquitin chain length and topology. Carvalho, A. High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.
Bremm, A. Lyslinked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Synthesis and analysis of Klinked ubiquitin chains. Methods Mol. Michel, M. Assembly and specific recognition of K and Klinked polyubiquitin. Cell 58 , 95— Chen, Z. A kilodalton ubiquitin carrier protein E2 catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. Haldeman, M. Structure and function of ubiquitin conjugating enzyme EK: the tail is a core-dependent activity element.
Biochemistry 36 , — Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair.
Cell 96 , — Sone, T. Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae. The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48 , — Characterization of the proteasome using native gel electrophoresis. Download references. You can also search for this author in PubMed Google Scholar.
Correspondence to Daniel Finley or Andreas Matouschek. The other authors declare no competing interests. Peer review information Nature Communications thanks Yasushi Saeki and the other, anonymous, reviewer s for their contribution to the peer review of this work.
Peer reviewer reports are available. Reprints and Permissions. The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates. Nat Commun 11, Download citation. Received : 02 October Accepted : 20 November Published : 24 January Anyone you share the following link with will be able to read this content:.
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Cell Discovery Nature Communications By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Enzyme mechanisms Proteasome. Abstract Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure.
Introduction Regulated protein degradation in eukaryotic cells is primarily mediated by the ubiquitin proteasome system. Full size image. Results Proteasome substrates with defined polyubiquitin chains We created substrates with ubiquitin chains of defined lengths and linkages attached to a base protein 24 Supplementary Fig. Substrates with Klinked ubiquitin chains We first asked how the proteasome recognizes proteins targeted for proteolysis by the canonical degradation signal as defined originally by Pickart and colleagues 5 , which is formed by a polyubiquitin chain consisting of four or more ubiquitin molecules linked through K Discussion Ubiquitin can form chains of different lengths and linkages and modify proteins at one or more positions.
Protein expression and purification Ub and Ub 4 M1 , and Ub K48R were purified using established protocols 24 , 86 , Generating ubiquitin chains Ub 4 K48 , Ub 4 K63 , Ub 8 K48 , and Ub 4 K11 ubiquitin chains were generated using enzymes that are part of the natural synthesis machinery and purified following published protocols 24 , 87 as described below. Generation of ubiquitinated substrate Ubiquitinated substrates were generated following published protocols Proteasome purification Proteasome was purified from S.
Kinetic plate reader assays Single-turnover degradation assays were performed as previously described Native proteasome gels Proteasome reconstitutions were resolved on 3. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability The source data underlying Figs. The cells in your body build up and break down proteins at a rapid rate. Ubiquitin attaches to proteins, tagging them for disposal.
This process is called ubiquitination. Tagged proteins are taken to proteasomes to be destroyed. Just before the protein enters the proteasome, ubiquitin is disconnected to be used again. In , the Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose for the discovery of this process, called ubiquitin mediated degradation proteolysis. Based on its function, ubiquitin has been studied for a role in potential targeted therapy to treat cancer.
Doctors focus on specific irregularities in the cancer cells that allow them to survive. The goal is to use ubiquitin to manipulate the protein in cancer cells to cause the cancer cell to die. The study of ubiquitin has led to the development of three proteasome inhibitors approved by the Food and Drug Administration FDA to treat people with multiple myeloma , a form of blood cancer:.
According to the National Cancer Institute , researchers are studying ubiquitin in relationship to normal physiology, cardiovascular disease , cancer , and other disorders. Ubiquitin plays an important role in regulating protein on the cellular level. Doctors believe it has promising potential for a variety of targeted cellular medicine treatments. The study of ubiquitin has already led to the development of medications for the treatment of multiple myeloma, a form of blood cancer.
These medications include bortezomib Velcade , carfilzomib Kyprolis , and ixazomib Ninlaro. Proteolytic enzymes perform many functions in your body. It is noteworthy that HUWE is overexpressed in lung, breast and colon carcinoma, suggesting also a possible role in tumorigenesis Adhikary et al. Fluorescence-activated cell sorting FACS was employed to obtain a population enriched for cells with elevated GFP-CL1 levels, which were subsequently analyzed by barcode sequencing Leto et al.
In a different screen, the near-haploid cell line KBM7 Carette et al. In a UPS-specific and genome-wide siRNA-based screen, a fluorescently tagged, thermally unstable nuclear reporter was used to identify proteins involved in nuclear protein quality control Pegoraro et al. The screen was performed by analyzing well plates with automated fluorescence microscopy. Besides a number of hits that were anticipated, such as proteasome subunits, they found the proteasome assembly chaperone POMP Burri et al.
The performance of the library and setup of the screen was confirmed as shRNAs directed against proteasome subunits were readily identified by accumulation of the reporter substrate.
In this particular case, the UPS reporter were used as a fast and robust tool for validation of the screening libraries. With the appearance of advanced techniques and equipment, high-throughput and high-content screenings have become attractive approaches for addressing biological questions and drug development.
Many different fluorescent substrate reporters have emerged over the years in parallel with a better understanding of the UPS and an increased awareness of the UPS as therapeutic target.
A number of opportunities for optimizing these reporter assays and tailoring them to specific purposes remain. Although some UPS reporter mouse strains have been generated, it is obvious that mouse models are not suited for large-scale screening efforts.
However, other animal reporter models, such as nematodes Hamer et al. In addition to UPS inhibitors, there is an emerging interest for UPS stimulators, which may be harder to identify with the currently available assays.
Hence, there is a need for the development of novel reporters that are more suited for detecting an increase in UPS activity. A point of improvement may be the use of internal stable reference proteins, which have been already applied in some screens Yen and Elledge, ; Yen et al. These reference proteins can correct for differences in synthesis of the reporter and may give a more robust readout, thereby reducing the number of false hits.
Due to the relatively low steady-state levels of the presently available reporter substrates, detection of enhanced degradation in high content screens may be problematic, even in the presence of a stable reference protein.
Two recent studies elegantly overcame this limitation by creating a system in which the expression of the reporter protein is repressed by a transcriptional regulator Zhao et al. Fusion of the transcriptional repressor to a destabilizing signal results in an inverse correlation between the activity of the UPS and the levels of the reporter protein that are regulated by the repressor.
However, the applicability of these reporter systems in high-throughput campaigns remains to be validated. Genetic screens and compound screens each have their own strengths and weaknesses and can complement each other in screening campaigns. Genetic screens may result in the identification of interesting but poorly druggable candidates, while compound screens may identify powerful drug-like compounds but encounter difficulties in target identification.
Upon combination of these complementary approaches and adapting the assays to more disease-relevant settings, fluorescent reporters can be used to their full potential in the pursuit for novel ways of modulating the UPS in human diseases. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abbott, C. Translation factors: in sickness and in health.
Trends Biochem. Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 5, — Adhikary, S. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation.
Cell , — Bachmair, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science , — Bard, J. Structure and Function of the 26S Proteasome. Bence, N. Impairment of the ubiquitin-proteasome system by protein aggregation. Berner, N. Protein quality control of the endoplasmic reticulum and ubiquitin-proteasome-triggered degradation of aberrant proteins: yeast pioneers the path.
Boland, B. Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing. Bove, J. Proteasome inhibition and Parkinson's disease modeling. Bruning, A. Misfolded proteins: from little villains to little helpers in the fight against cancer.
Burri, L. Identification and characterization of a mammalian protein interacting with 20S proteasome precursors. Carette, J. Haploid genetic screens in human cells identify host factors used by pathogens. Chen, D. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res. Chou, T. Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system.
Ciechanover, A. Proteolysis: from the lysosome to ubiquitin and the proteasome. Cell Biol. Dantuma, N. Elsasser, S. Delivery of ubiquitinated substrates to protein-unfolding machines. Fiebiger, E. Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin. Cell 15, — Fricke, B. The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum.
EMBO Rep. Fricker, L. Proteasome inhibitor drugs. Gilon, T. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBO J. Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair. Hamer, G. A photoconvertible reporter of the ubiquitin-proteasome system in vivo. Methods
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