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Dss1 is a 26S proteasome ubiquitin receptor.

ABSTRACT: The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.

SUBMITTER: Paraskevopoulos K

PROVIDER: S-EPMC4232310 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Dss1 is a 26S proteasome ubiquitin receptor.

Paraskevopoulos Konstantinos K Kriegenburg Franziska F Tatham Michael H MH Rösner Heike I HI Medina Bethan B Larsen Ida B IB Brandstrup Rikke R Hardwick Kevin G KG Hay Ronald T RT Kragelund Birthe B BB Hartmann-Petersen Rasmus R Gordon Colin C

Molecular cell 20141009 3

The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and ...[more]

PMID: 25306921

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Project description:The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities.

Project description:BackgroundUbiquitination is a post-translational modification where ubiquitin is covalently attached to lysine residues on substrate proteins to signal their degradation by the 26S proteasome or initiate other non-degradation functions such as cellular trafficking. The diversity of ubiquitin modifications can be attributed to the variable number of ubiquitin molecules attached to a lysine residue (mono- vs. poly-ubiquitin chains), the type of covalent linkages within poly-ubiquitin chains and the number of lysine residues on a substrate that are occupied by ubiquitin at any given time. The integral role ubiquitination plays in cell homeostasis is reflected by the multitude of diseases associated with impaired ubiquitin modification, rendering it the focus of extensive research initiatives and proteomic discovery studies. However, determining the functional role of distinct ubiquitin modifications directly from proteomic data remains challenging and represents a bottleneck in the process of deciphering how ubiquitination at specific substrate sites impacts cell signaling.MethodsIn this study SILAC coupled with LC-MS/MS is used to identify ubiquitinated proteins in SKOV3 ovarian cancer cells, with the implementation of a computational approach that measures relative ubiquitin occupancy at distinct modification sites upon 26S proteasome inhibition and uses that data to infer functional significance.ResultsIn addition to identifying and quantifying relative ubiquitin occupancy at distinct post-translational modification sites to distinguish degradation from non-degradation signaling, this research led to the discovery of nine ubiquitination sites in the oncoprotein HER2 that have not been previously reported in ovarian cancer. Subsequently the computational approach applied in this study was utilized to infer the functional role of individual HER2 ubiquitin-modified residues.ConclusionsIn summary, the computational method, previously described for glycosylation analysis, was used in this study for the assessment of ubiquitin stoichiometries and applied directly to proteomic data to distinguish degradation from non-degradation ubiquitin functions.

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Dss1 is a 26S proteasome ubiquitin receptor.

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Dss1 is a 26S proteasome ubiquitin receptor.

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