rpn13

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1 , show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant...

UNLABELLED: Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1 , show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant...

Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy, and its specific pathological mechanism remains to be fully clarified. Adhesion-regulating molecule 1 (ADRM1) has been proven to be involved in OA progression as a favorable gene. However, the exact mechanism of ADRM1 involved in OA were unknown. Here, we showed that the ADRM1 expression decreased in human OA cartilage, destabilization of the medial meniscus (DMM)-induced mouse OA cartilage, and interleukin (IL)-1β-induced primary mouse articular chondrocytes...

Bortezomib has been successful for treatment of multiple myeloma, but not against solid tumors, and toxicities of neuropathy, thrombocytopenia and the emergence of resistance have triggered efforts to find alternative proteasome inhibitors. Bis-benzylidine piperidones such as RA190 covalently bind ADRM1/RPN13, a ubiquitin receptor that supports recognition of polyubiquitinated substrates of the proteasome and their subsequent deububiqutination and degradation. While these candidate RPN13 inhibitors (iRPN13) show promising anticancer activity in mouse models of cancer, they have suboptimal drug-like properties...

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site...

Bortezomib and the other licensed 20S proteasome inhibitors show robust activity against liquid tumors like multiple myeloma, but have disappointed against solid tumors including ovarian cancer. Consequently, interest is mounting in alternative non-peptide based drugs targeting the proteasome's 19S regulatory particle subunit, including its ubiquitin receptor RPN13. RA183 and RA375 are more potent analogs of the prototypic inhibitor of RPN13 (iRPN13) called RA190, and they show promise for the treatment of ovarian cancer...

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305)...

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The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains...

The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome...

Ubiquitin like protein 5 (UBL5) interacts with other proteins to regulate their function but differs from ubiquitin and other UBLs because it does not form covalent conjugates. Ubiquitin and most UBLs mediate the degradation of target proteins through the 26S proteasome but it is not known if UBL5 can also do that. Here we found that the UBL5s of rice and Nicotiana benthamiana interacted with rice stripe virus (RSV) p3 protein. Silencing of NbUBL5s in N. benthamiana facilitated RSV infection, while UBL5 overexpression conferred resistance to RSV in both N...

We previously reported a peptoid ligand for the proteasomal ubiquitin receptor Rpn13 called KDT-11 and demonstrated that this compound is toxic to multiple myeloma cells, but not non-malignant cells. Here, we show that KDT-11 decreases the viability of a variety of cancer cell lines, especially melanomas and various blood cancers. The peptoid induces selective G1 cell-cycle arrest, resulting in eventual apoptosis. While KDT-11 does not antagonize any of the known protein-protein interactions involving Rpn13, the peptoid inhibits the ability of Rpn13 to stimulate the activity of an associated deubiquitylase Uch37/UCHL5 in vitro, suggesting a high level of Uch37 activity might be important for cancer cell proliferation...

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue...

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death...

Good evidence exists that the ubiquitin-proteasome system (UPS) plays an important role in degradation of mitochondrial proteins and membrane proteins associated with mitochondria (MAM proteins). Mitochondria contain all components of the ubiquitin-conjugating system, which are necessary for the attachment of ubiquitin molecules to target proteins, subjected to subsequent degradation in proteasomes. An important stage in the delivery of proteins for proteolytic degradation in proteasomes is their interaction with ubiquitin receptors located on the regulatory subunit (19S) of the proteasome: the Rpn10 or Rpn13 subunit...

BACKGROUND: According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC...

Rpn13/Adrm1 is recruited to the proteasome by PSMD1/Rpn2, where it serves as a substrate receptor that binds preferentially to K48-linked ubiquitin chains, an established signal for protein proteolysis. Here, we use NMR to solve the structure of hRpn13 Pru:hRpn2 (940-953):K48-diubiquitin. Surprisingly, hRpn2-bound hRpn13 selects a dynamic, extended conformation of K48-diubiquitin that is unique from previously determined structures. NMR experiments on free K48-diubiquitin demonstrate the presence of the reported "closed" conformation observed by crystallography, but also this more extended state, in which the hRpn13-binding surface is exposed...

Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure. Three proteasome subunits-Rpn10, Rpn13, and Rpn1-can recognize ubiquitin chains. Here we report that proteins with single chains of K48-linked ubiquitin are targeted for degradation almost exclusively through binding to Rpn10. Rpn1 can act as a co-receptor with Rpn10 for K63 chains and for certain other chain types. Differences in targeting do not correlate with chain affinity to receptors...

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic...

The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain...