Proteasomal Ubiquitin Receptor Research Articles

A viral movement protein targets host catalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat

The infection of host plants by many different viruses causes reactive oxygen species (ROS) accumulation and yellowing symptoms, but the mechanisms through which plant viruses counteract ROS-mediated immunity to facilitate infection and symptom development have not been fully elucidated. Most plant viruses are transmitted by insect vectors in the field, but the molecular mechanisms underlying virus‒host–insect interactions are unclear. In this study, we investigated the interactions among wheat, barley yellow dwarf virus (BYDV), and its aphid vector and found that the BYDV movement protein (MP) interacts with both wheat catalases (CATs) and the 26S proteasome ubiquitin receptor non-ATPase regulatory subunit 2 homolog (PSMD2) to facilitate the 26S proteasome-mediated degradation of CATs, promoting viral infection, disease symptom development, and aphid transmission. Overexpression of the BYDV MP gene in wheat enhanced the degradation of CATs, which leading to increased accumulation of ROS and thereby enhanced viral infection. Interestingly, transgenic wheat lines overexpressing BYDV MP showed significantly reduced proliferation of wingless aphids and an increased number of winged aphids. Consistent with this observation, silencing of CAT genes also enhanced viral accumulation and reduced the proliferation of wingless aphids but increased the occurrence of winged aphids. In contrast, transgenic wheat plants overexpressing TaCAT1 exhibited the opposite changes and showed increases in grain size and weight upon infection with BYDV. Biochemical assays demonstrated that BYDV MP interacts with PSMD2 and promotes 26S proteasome-mediated degradation of TaCAT1 likely in a ubiquitination-independent manner. Collectively, our study reveals a molecular mechanism by which a plant virus manipulates the ROS production system of host plants to facilitate viral infection and transmission, shedding new light on the sophisticated interactions among viruses, host plants, and insect vectors.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two β-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

Abstract 4941: Development of small molecule RPN13 inhibitors for treatment of glioblastoma

Abstract Glioblastoma (GBM) is an aggressive and deadly brain cancer with a one-year median survival due to the lack of effective targeted treatments. Our goal is to develop a small molecule inhibitor targeting RPN13 protein in the 19S proteasome complex as a new drug for GBM. RPN13 is a proteasome ubiquitin receptor that recognizes ubiquitin-conjugated proteins that are thus tagged for degradation via the 20S proteasome enzymatic complex. GBM is highly sensitive to proteasome inhibition due to its aggressive growth and accumulation of misfolded polyubiquitinated proteins that is insufficiently managed despite increased proteasome levels and enhanced proteasome activity (i.e. proteotoxic stress). Up Therapeutics judiciously designed drug-like RPN13 inhibitors to overcome limitations in the prototype RPN13 inhibitor RA190 identified at Johns Hopkins University. We selected Up284 as lead compound based upon our preliminary studies showing drug-like characteristics, specificity (binding to Cys88 of RPN13), and proteasome inhibition that produces a cytotoxic accumulation of polyUb proteins, ER stress, and reactive oxygen species. Up284 treatment selectively triggers apoptosis in solid tumor types including GBM cell lines and oncospheres. A suitable Up284 formulation was selected by measuring aqueous solubility, stability and on-target activity in mice imaged using a proteasome-dependent in vivo reporter. Up284 can be administered i.v. or orally, and it penetrates through the BBB. As a proof of concept for treatment of GBM, Up284 demonstrated on-target activity in intracranial syngeneic GL261 tumor model by inhibiting proteasome function in tumor tissues. Up284 also well tolerated and regressed tumors in syngeneic, xenograft and spontaneous models of ovarian and breast cancers with acceptable safety profile. Up284 also synergize with current frontline chemotherapeutics and can be used as a single agent or in combination for the treatment of wide range of cancers. Citation Format: Yung-Nien Chang, Ravi K. Anchoori, Alex Shimura Yamashita, Marina da Costa Rosa. Development of small molecule RPN13 inhibitors for treatment of glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4941.

HMG20A was identified as a key enhancer driver associated with DNA damage repair in oral squamous cell carcinomas

BackgroundOral squamous cell carcinoma (OSCC) is the main type of oral cancer. Disturbing DNA repair is an invaluable way to improve the effectiveness of tumor treatment. Here, we aimed to explore the key enhancer drivers associated with DNA damage repair in OSCC cells.MethodsGene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA) and Kaplan-Meier analysis were applied to explore the relationship among DNA repair-related genes expression and clinical phenotypes based on The Cancer Genome Atlas (TCGA) database. HOMER software and Integrative Genomics Viewer were applied to identify and visualize enhancers using GSE120634. Toolkit for Cistrome Data Browser was applied to predict transcription factors. Human Protein Atlas Database was used to analyze the protein levels of transcription factors in OSCC and control tissues. Seventy-two OSCC patients were included in this study. qRT-PCR was used to detect transcription factor expression in OSCC and adjacent control tissues collected in this study. qRT-PCR and ChIP-qPCR were used to verify the binding of transcription factors to enhancers, and regulation of target genes transcription. Transcription factor knockdown and control cells were treated with cisplatin. CCK8 was used to detect cell viability and proliferation. Western blotting was implemented to detect the levels of DNA repair-related proteins. Transwell assay was used to detect cell invasion.ResultsDNA repair was positively associated with the OSCC metastatic phenotype. Patients in the cluster with high expression of DNA repair-related genes had a worse prognosis and a higher proportion of advanced stage, low-differentiation, alcohol consumption and smoking compared to the cluster with low DNA repair-related gene expression. Seventeen metastasis-specific enhancer-controlled upregulated DNA repair-related genes, with the top two upregulated genes being ADRM1 26 S proteasome ubiquitin receptor (ADRM1) and solute carrier family 12 member 7 (SLC12A7) were screened. High mobility group 20 A (HMG20A) was the key prognostic enhancer driver regulating metastasis-specific DNA repair-related genes, with higher expression in OSCC tissues than normal control tissues, and higher expression in metastatic OSCC tissues than non-metastatic OSCC tissues. HMG20A bound to the metastasis-specific enhancers of ADRM1 and SLC12A7, thereby promoting ADRM1 and SLC12A7 expression. Knockdown of HMG20A enhanced cisplatin sensitivity of cells, and inhibited OSCC cells from repairing DNA damage caused by cisplatin, as well as proliferation and invasion of OSCC cells.ConclusionHMG20A was identified as the key prognostic enhancer driver regulating DNA repair in OSCC cells, providing a new therapeutic target for OSCC.

Abstract 1781: Up284, a novel inhibitor to treat quadruple negative breast cancer

Abstract Quadruple negative breast cancer (QNBC), lacking the expression of ER (estrogen receptor), PR (progesterone receptor), HER2 (human epidermal growth factor receptor 2) and AR (androgen receptor), is the breast cancer subtype with the worst prognosis, and QNBC disproportionately afflicts African Americans. It has no standard-of-care treatment targets and thus efficacious and safe treatments are urgently sought for this unmet medical need, and to address the disparity in breast cancer outcomes. The current proposal is motivated by data showing elevated expression of proteasome subunit RPN13 is associated with both African American race and lower survival in QNBC patients, that RPN13 selectively targeted by a small molecule Up284 developed by Up Therapeutics. Both triple negative breast cancer and QNBC cell lines show evidence of greater vulnerability to proteasome inhibitors. However, licensed 20S proteasome inhibitors, e.g. bortezomib, have proven ineffective against solid tumors, with emergence of resistance, and dose limiting toxicities including thrombocytopenia and neutropenia. Up284 has a target and structure designed to overcome the limitations of the licensed drugs with respect to drug resistance. Up284 blocks substrate recognition and deubiquitination rather than just one of the three 20S catalytic activities), poor activity against solid tumors (Up284 has a novel spiro structure with evidence of improved drug access to tumor as compared to peptide-based 20S inhibitors), key toxicities of thrombocytopenia and neutropenia (unlike 20S inhibitors, Up284 does not target the immunoproteasome expressed by hematopoietic cells and does not show these toxicities). Up284 shows broad anticancer activity in vitro, including against QNBC lines with a robust therapeutic index, a promising safety profile and pharmacodynamics, and the ability to control xenograft tumor. By inhibiting proteasome ubiquitin receptor RPN13 function and its associated deubiquitinase activity, Up284 triggers more rapid accumulation and increased molecular weight polyubiquinated protein aggregates than is induced by 20S inhibitors. These toxic misfolded protein aggregates produce an unresolved ER stress, activate the canonical Unfolded Protein Response (UPR) signaling cascade and more rapidly triggers apoptosis than 20S inhibitor. The safety parameters and promising efficacy of Up284 against breast cancer lines encourages us to validate Up284 efficacy in more QNBC lines and animal models. We discuss Up284 efficacy and pharmacological analysis in our presentation. Citation Format: Balasubramanyam Karanam, Clayton Yates, Ravi Anchoori. Up284, a novel inhibitor to treat quadruple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1781.

Targeting Proteasome- Associated Ubiquitin Receptor PSMD4/Rpn10 in Multiple Myeloma

The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MABackground and Rationale Normal cellular homeostasis is maintained by a balanced regulation of protein synthesis and degradation via the ubiquitin proteasome system (UPS). Deregulation of the UPS is linked to pathogenesis of various human diseases, including cancer. Targeting the 20S proteasome in the UPS via proteasome inhibitors bortezomib, carfilzomib, or ixazomib has proven effective therapy for relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit their long-term utility. Therapeutic strategies directed against ubiquitin receptors upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Ubiquitin-tagged proteins are recognized by 19S proteasome-associated ubiquitin receptors; the ubiquitin moiety is then removed from the substrate protein via deubiquitylating (DUB) enzyme enzymatic activity; target protein is then unfolded by the AAA-ATPases; followed by degradation of proteins through 20S proteolytic activities. Here we examined the role of ubiquitin receptor Rpn10/PSMD4 in MM using RNA interference strategies.Materials and Methods We utilized MM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) or Rpn10-siRNA ON TARGET plus SMART pool using the cell line Nucleofector Kit V. Signal transduction pathways were evaluated using immunoblotting. Cell viability were assessed using WST-1 assay. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student's t test.Results1) A statistically significant inverse correlation was noted between Rpn10 levels and overall patient survival (n=175) (p= 0.00064). 2) Analysis of PSMD4/Rpn10 gene expression showed a significantly higher level in primary patient MM cells versus normal plasma cells or PBMCs (p = 0.023 or p = 0.001, respectively). 3) Rpn10 protein is more highly expressed in MM cells than in normal cells. 4) Functional studies showed that blockade of Rpn10 using RNA interference (siRNA and shRNA) significantly decreased MM cell viability (p < 0.001; n=3). 5) Rpn10-shRNA knockdown decreased MM cell proliferation. These findings were further confirmed in doxycycline-inducible Rpn10-shRNA knockout cells. 6) Transfection of Rpn10-siRNA in MM cells increased accumulation of poly-ubiquitylated proteins. 7) To test whether Rpn10 knockdown blocks cellular proteasome function, we used a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Rpn10-siRNA increased accumulation of the Ub-GFP reporter indicating impaired proteasome degradation. 8) Mechanistic studies show that Rpn10-siRNA-triggered MM cell death is associated with: (i) cell cycle arrest and apoptosis; (ii) increase in poly-ubiquitylated proteins; and (iii) activation of caspases and induction of endoplasmic stress response signaling.Conclusion Our preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of proteasome in the ubiquitin proteasomal cascade, and provides the framework for development of proof-of-concept inhibitors targeting Rpn10 for future evaluation in clinical trials. DisclosuresChauhan:Triphase accelerator: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder.

Mechanistic Studies of the Multiple Myeloma and Melanoma Cell-Selective Toxicity of the Rpn13-Binding Peptoid KDT-11

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 invitro, suggesting a high level of Uch37 activity might be important for cancer cell proliferation. However, a variety of experiments in SK-MEL-5 melanoma cells suggest that KDT-11's cytotoxic effects are mediated by interactions with proteins other than Rpn13.

The Extent of Extended-Ubiquitin Binding to the Proteasome

In this issue of Structure, Lu etal. (2020) describe an NMR-based study showing the proteasome ubiquitin receptor hRpn13 bound to an extended conformation of K48-diubiquitin that is different from previously described structures of K48-diubiquitin. Observed dynamic binding properties suggest an ability of substrates to hop between proteasome ubiquitin receptors.

K48 Linked Ubiquitin Chains Affect Proteasomal Unfolding Ability via Interactions with Rpn10

The ubiquitin proteasome system plays a crucial role in homeostasis by preferentially degrading proteins that are damaged, misfolded, or no longer necessary in regulatory processes. Substrates are recognized and polyubiquitinated by E1, E2, and E3 enzymes, which leads to recruitment and degradation by the proteasome. However, the proteasome does not always completely degrade its target and will release a protein fragment if unfolding does not occur. We previously showed that the proteasome’s unfolding ability is dependent on the ubiquitin chains’ interactions with proteasomal ubiquitin receptors, and that different chain architectures (linear K63 chains vs branched K48 and K63‐containing chains) had different receptor dependencies. To investigate the interaction of substrates bearing linear K48‐linked chains, we turned to the E3 ligase Ubr1, which recognizes N‐end rule substrates. A model unfolding ability substrate containing an N‐terminal arginine was degraded by wild‐type and ubiquitin‐receptor mutant proteins. The results indicate that although ubiquitin receptors Rpn1, Rpn10, and Rpn13 are all important for degradation of this substrate, Rpn10 has the greatest effect on the proteasome’s unfolding ability.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.

Study of Humoral Responses against Lomentospora/Scedosporium spp. and Aspergillus fumigatus to Identify L. prolificans Antigens of Interest for Diagnosis and Treatment.

The high mortality rates of Lomentospora prolificans infections are due, above all, to the tendency of the fungus to infect weakened hosts, late diagnosis and a lack of effective therapeutic treatments. To identify proteins of significance for diagnosis, therapy or prophylaxis, immunoproteomics-based studies are especially important. Consequently, in this study murine disseminated infections were carried out using L. prolificans, Scedosporium aurantiacum, Scedosporium boydii and Aspergillus fumigatus, and their sera used to identify the most immunoreactive proteins of L. prolificans total extract and secreted proteins. The results showed that L. prolificans was the most virulent species and its infections were characterized by a high fungal load in several organs, including the brain. The proteomics study showed a high cross-reactivity between Scedosporium/Lomentospora species, but not with A. fumigatus. Among the antigens identified were, proteasomal ubiquitin receptor, carboxypeptidase, Vps28, HAD-like hydrolase, GH16, cerato-platanin and a protein of unknown function that showed no or low homology with humans. Finally, Hsp70 deserves a special mention as it was the main antigen recognized by Scedosporium/Lomentospora species in both secretome and total extract. In conclusion, this study identifies antigens of L. prolificans that can be considered as potential candidates for use in diagnosis and as therapeutic targets and the production of vaccines.

Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes

The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding or aggregation and the cellular proteostasis network that monitors and maintains proteome health is poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear. Because aggregation in cells is rapid, nonlinear, and asynchronous, it has not been possible to deconvolve these kinetically linked processes to determine the earliest cellular responses to misfolded proteins. Upon removal of the synthetic, biologically inert ligand shield-1 (S1), AgDD, an engineered variant FK506-binding protein (FKBP1A), rapidly (t½ ∼5 min) unfolds and self-associates, forming detergent-insoluble, microscopic cytoplasmic aggregates. Using global diglycine-capture (K-GG) proteomics, we found here that this solubility transition is associated with immediate increases in ubiquitylation of AgDD itself, along with that of endogenous proteins that are components of the ribosome and the 26S proteasome. We also found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1). We conclude that these proteasomal responses are due to AgDD protein misfolding and not to the presence of detergent-insoluble aggregates.

Ubiquitin Subproteome of Brain Mitochondria and Its Changes Induced by Experimental Parkinsonism and Action of Neuroprotectors.

The review summarizes the data of our research and published studies on the ubiquitination of brain mitochondrial proteins and its changes during the development of experimental parkinsonism and administration of the neuroprotector isatin (indole-2,3-dione) with special attention to the mitochondrial ubiquitin-conjugating system and location of ubiquitinated proteins in these organelles. Incubation of brain mitochondrial fraction with biotinylated ubiquitin in vitro resulted in the incorporation of biotinylated ubiquitin in both mitochondrial and mitochondria-associated proteins. According to the interactome analysis, the identified non-ubiquitinated proteins are able to form tight complexes with ubiquitinated proteins or their partners and components of mitochondrial membranes, in which interactions of ubiquitin chains with the ubiquitin-binding protein domains play an important role. The studies of endogenous ubiquitination in the total brain mitochondrial fraction of C57Bl mice performed in different laboratories have shown that mitochondrial proteins represent about 30% of all ubiquitinated proteins. However, comparison of brain subproteomes of mitochondrial ubiquitinated proteins reported in the literature revealed significant differences both in their composition and involvement of identified ubiquitinated proteins in biological processes listed in the Gene Ontology database. The development of experimental parkinsonism in C57Bl mice induced by a single-dose administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a decrease in the total number of mitochondrial ubiquitinated proteins and increase in the number of oxidized mitochondrial proteins containing the ubiquitin signature (K-ε-GG). Comparison of ubiquitinated proteins associated with the mouse brain mitochondrial fraction and mouse brain mitochondrial proteins bound to the proteasome ubiquitin receptor (Rpn10 subunit) did not reveal any common proteins. This suggests that ubiquitination of brain mitochondrial proteins is not directly related to their degradation in the proteasomes. Proteomic profiling of brain isatin-binding proteins identified enzymes involved in the ubiquitin-conjugating system functioning. Mapping of the identified isatin-binding proteins to known metabolic pathways indicates their participation in the parkin (E3 ubiquitin ligase)-associated pathway (CH000000947). The functional links involving brain mitochondrial ubiquitinated proteins were found only in the group of animals with the MPTP-induced parkinsonism, but not in animals treated with MPTP/isatin or isatin only. This suggests that the neuroprotective effect of isatin may be associated with the impaired functional relationships of proteins targeted to subsequent degradation.

Ubiquitin receptors are required for substrate-mediated activation of the proteasome\u2019s unfolding ability

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. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

PSMD4 regulates the malignancy of esophageal cancer cells by suppressing endoplasmic reticulum stress.

Proteasome 26S subunit non-ATPase 4 (PSMD4) is an important proteasome ubiquitin receptor and plays a key role in endoplasmic reticulum stress (ERS). However, the study of PSMD4 in esophageal cancer (EC) is relatively rare. Here, we found that the expression of PSMD4 was markedly enhanced in EC tissues and cell lines. The cell counting kit-8 (CCK-8) assay showed that overexpression of PSMD4 significantly enhanced Eca109 cell viability, while inhibition of PSMD4 reduced Eca109 cell viability. Knockdown of PSMD4 induced Eca109 cell apoptosis and cell cycle arrest. More importantly, knockdown of PSMD4 significantly enhanced the expression of glucose regulated protein 78, activating transcription factor 6, and p-protein kinase R-like ER kinase, indicating an enhanced ERS response in esophageal cancer cells. Compared with the control cells, brefeldin A significantly inhibited the expression of PSMD4 and increased the expression of p53-upregulated modulator of apoptosis. However, such effects were largely reversed after overexpressing PSMD4 in Eca109 cells, suggesting that silencing PSMD4 could enhance ERS-induced cell apoptosis. In summary, upregulation of PSMD4 promoted the progression of esophageal cancer mainly by reducing ERS-induced cell apoptosis.

Blockade of Ubiquitin Receptor PSMD4/Rpn10 Triggers Cytotoxicity and Overcomes Bortezomib-Resistance in Multiple Myeloma

Background and Rationale Proteasome inhibitors (PIs) are standard of care therapy for patients diagnosed with multiple myeloma (MM). However, prolonged treatment can be associated with toxicity and development of drug resistance. Our research efforts have therefore focused on designing therapeutic strategies that can overcome PI-resistance. In this context, our RNA-interference-based loss-of-function studies have identified 19S proteasome-associated Ubiquitin Receptor (UbRs) Rpn10 and Rpn13 as potential therapeutic targets. Both Rpn10 and Rpn13 UbRs are associated with the 19S regulatory lid of the proteasome that recognizes ubiquitylated proteins marked for degradation by 20S core particle. We have previously reported the role of Rpn13 in MM (Song et., al Luekemia 2016 Sep;30(9):1877-86). Here we functionally characterized the role of Rpn10 in MM.Materials and Methods Cytotoxicity assays were performed using a panel of MM cell lines, primary patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST-1, MTT, and Cell Titer-Glo assay.Signal transduction pathways were evaluated using immunoblotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Statistical significance of data was determined using a Student's t test.Results Functional characterization of Rpn10 in MM:Gene expression (GEP) analysis showed inverse correlation between Rpn10 and overall patient survival (n=175) (p= 0.00064). Immunoblot analysis showed high Rpn10 protein levels in primary patient cells and MM cell lines versus normal plasma cells or PBMCs. Rpn10 knockdown by siRNA significantly decreased MM cell viability in both bortezomib-sensitive and -resistant MM cell lines. To assess the Rpn10 function, we generated a doxycycline-inducible Rpn10-shRNA knockout stable MM cell line. Rpn10-shRNA knockdown decreased MM cell proliferation. Mechanistic studies show that Rpn10 knockdown-triggered MM cell death is associated with 1) accumulation of cells in early and late apoptotic phase; 2) increase in polyubiquinated proteins; 3) arrest of cell cycle; 4) induction of ER stress; and 5) activation of caspases mediating apoptotic pathways. In order to determine if blockade of Rpn10 affects cellular proteasome function, we next utilized a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Interestingly, Rpn10-siRNA increased accumulation of the Ub-GFP reporter, reflecting impaired proteasome-mediated protein degradation. Finally, treatment of MM cells with peptides targeting UIM2 domain of Rpn10 significantly decreased MM cell viability, suggesting a potential therapeutic approach in MM.ConclusionOur preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of the proteasome in the ubiquitin proteasomal cascade, and provides the framework for clinical evaluation of Rpn10 inhibitors to overcome PI resistance and improve patient outcome in MM. DisclosuresAnderson:C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Consultancy; OncoPep: Equity Ownership, Other: Scientific founder.

Biochemical properties of K11,48-branched ubiquitin chains

As one of the most widely existing post-translational modification models, ubiquitination regulates diverse cellular activities. In eukaryotes, K11,48-branched ubiquitin chains play key roles in cell cycle and protein quality control. However, the structural and biochemical properties of K11,48-branched ubiquitin chains have not been well examined. Here we employed the synthetic linkage- and length-defined K11,48-branched ubiquitin chains to examine their binding and hydrolysis properties in vitro. Quantitatively affinity determination of ubiquitin chains to the proteasome ubiquitin receptor S5a indicated that the S5a exhibited preference binding to K11,48-branched chains over K11-linked chains, but not K48-conjugated chains. In addition, deubiquitination experiments were carried out and the results showed that K11,48-branched chains were preferably hydrolyzed by proteasome-associated deubiquitinase Rpn11 than homotypic K11 or K48-linked chains.

Semi-synthesis of disulfide-linked branched tri-ubiquitin mimics

Ubiquitin (Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub (Ub311/48(S–S)), the first example of an isopeptide mimic for the branched Ub chains, which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the E1-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11C, K48C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub311/48(S–S). Deubiquitination and pulldown assays indicated that the synthetic Ub311/48(S–S) could be hydrolyzed by linkage-specific deubiquitinases (K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5a.

Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry.

Protein-protein interactions are essential for protein complex formation and function. Affinity purification coupled with mass spectrometry (AP-MS) is the method of choice for studying protein-protein interactions at the systems level under different physiological conditions. Although effective in capturing stable protein interactions, transient, weak, and/or dynamic interactors are often lost due to extended procedures during conventional AP-MS experiments. To circumvent this problem, we have recently developed XAP (in vivo cross-linking (X)-assisted affinity purification)-MS strategy to better preserve dynamic protein complexes under native lysis conditions. In addition, we have developed XBAP (in vivo cross-linking (X)-assisted bimolecular tandem affinity purification)-MS method by incorporating XAP with bimolecular affinity purification to define dynamic and heterogeneous protein subcomplexes. Here we describe general experimental protocols of XAP- and XBAP-MS to study dynamic protein complexes and their subcomplexes, respectively. Specifically, we present their applications in capturing and identifying proteasome dynamic interactors and ubiquitin receptor (UbR)-proteasome subcomplexes.

Quantitative Affinity Interaction of Ubiquitinated and Non-ubiquitinated Proteins with Proteasome Subunit Rpn10.

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (Kd= 2.4·10-6M) and pyruvate kinase (Kd= 2.8·10-5M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10-8and 3.2·10-9M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.

SQSTM1/p62-mediated autophagy compensates for loss of proteasome polyubiquitin recruiting capacity

ABSTRACTProtein homeostasis in eukaryotic cells is regulated by 2 highly conserved degradative pathways, the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy. Recent studies revealed a coordinated and complementary crosstalk between these systems that becomes critical under proteostatic stress. Under physiological conditions, however, the molecular crosstalk between these 2 pathways is still far from clear. Here we describe a cellular model of proteasomal substrate accumulation due to the combined knockdown of PSMD4/S5a and ADRM1, the 2 proteasomal ubiquitin receptors. This model reveals a compensatory autophagic pathway, mediated by a SQSTM1/p62-dependent clearance of accumulated polyubiquitinated proteins. In addition to mediating the sequestration of ubiquitinated cargos into phagophores, the precursors to autophagosomes, SQSTM1 is also important for polyubiquitinated aggregate formation upon proteasomal inhibition. Finally, we demonstrate that the concomitant stabilization of steady-state levels of ATF4, a rapidly degraded transcription factor, mediates SQSTM1 upregulation. These findings provide new insight into the molecular mechanisms by which selective autophagy is regulated in response to proteasomal overflow.