Receptor Ubiquitination Research Articles

The zinc-finger proteins WOC and ROW play distinct functions within the HP1c transcription complex

In Drosophila, the Heterochromatin Protein 1c (HP1c) forms a transcriptional complex with the zinc-finger proteins WOC and ROW, and the extraproteasomal ubiquitin receptor Dsk2. This complex localizes at promoters of active genes and it is required for transcription. The functions played by the different components of the HP1c complex are not fully understood. In this study we show that WOC and ROW are required for chromatin binding of both Dsk2 and HP1c. However, while impairing chromatin binding strongly destabilizes HP1c, it does not affect Dsk2 stability. We also show that WOC, but not ROW, is required for nuclear localization of Dsk2. Moreover, WOC and Dsk2 co-immunoprecitate upon ROW depletion. These results suggest that WOC and Dsk2 interact to form a subcomplex that mediates nuclear translocation of Dsk2. We also show that ROW mediates chromatin binding of the WOC/Dsk2 subcomplex, as well as of HP1c. Altogether these observations favor a model by which the interaction with WOC recruits Dsk2 to the HP1c complex that, in its turn, binds chromatin in a ROW-dependent manner.

The proteasome 19S cap and its ubiquitin receptors provide a versatile recognition platform for substrates

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. Surprisingly, in steady-state assays Rpn13 retarded degradation of various single-chain substrates. Substrates with multiple short ubiquitin chains can be presented for degradation by any of the known receptors, whereas those targeted to the proteasome through a ubiquitin-like domain are degraded most efficiently when bound by Rpn13 or Rpn1. Thus, the proteasome provides an unexpectedly versatile binding platform that can recognize substrates targeted for degradation by ubiquitin chains differing greatly in length and topology.

Microglial autophagy is impaired by prolonged exposure to β-amyloid peptides: evidence from experimental models and Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of misfolded proteins, amyloid-β (Aβ) aggregates, and neuroinflammation in the brain. Microglial cells are key players in the context of AD, being capable of releasing cytokines in response to Aβ and degrading aggregated proteins by mechanisms involving the ubiquitin-proteasome system and autophagy. Here, we present in vivo and in vitro evidence showing that microglial autophagy is affected during AD progression. PDAPPJ20 mice-murine model of AD-exhibited an accumulation of the autophagy receptor p62 and ubiquitin+ aggregates in Iba1+ microglial cells close to amyloid deposits in the hippocampus. Moreover, cultured microglial BV-2 cells showed an enhanced autophagic flux during a 2-h exposure to fibrillar Aβ, which was decreased if the exposure was prolonged to 24h, a condition analogous to the chronic exposure to Aβ in the human pathology. The autophagic impairment was also associated with lysosomal damage, depicted by membrane permeabilization as shown by the presence of the acid hydrolase cathepsin-D in cytoplasm and altered LysoTracker staining. These results are compatible with microglial exhaustion caused by pro-inflammatory conditions and persistent exposure to aggregated Aβ peptides. In addition, we found LC3-positive autophagic vesicles accumulated in phagocytic CD68+ microglia in human AD brain samples, suggesting defective autophagy in microglia of AD brain. Our results indicate that the capacity of microglia to degrade Aβ and potentially other proteins through autophagy may be negatively affected as the disease progresses. Preserving autophagy in microglia thus emerges as a promising approach for treating AD. Graphical abstract.

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

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. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.

Spatial Organization of Proteasome Aggregates in the Regulation of Proteasome Homeostasis.

Misfolded proteins and insoluble aggregates are continuously produced in the cell and can result in severe stress that threatens cellular fitness and viability if not managed effectively. Accordingly, organisms have evolved several protective protein quality control (PQC) machineries to address these threats. In eukaryotes, the ubiquitin–proteasome system (UPS) plays a vital role in the disposal of intracellular misfolded, damaged, or unneeded proteins. Although ubiquitin-mediated proteasomal degradation of many proteins plays a key role in the PQC system, cells must also dispose of the proteasomes themselves when their subunits are assembled improperly, or when they dysfunction under various conditions, e.g., as a result of genomic mutations, diverse stresses, or treatment with proteasome inhibitors. Here, we review recent studies that identified the regulatory pathways that mediate proteasomes sorting under various stress conditions, and the elimination of its dysfunctional subunits. Following inactivation of the 26S proteasome, UPS-mediated degradation of its own misassembled subunits is the favored disposal pathway. However, the cytosolic cell-compartment-specific aggregase, Hsp42 mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments, where they become extensively modified with ubiquitin, and are directed by ubiquitin receptors for autophagic clearance (proteaphagy). We also discuss the sorting mechanisms that the cell uses under nitrogen stress, and to distinguish between dysfunctional proteasome aggregates and proteasome storage granules (PSGs), reversible assemblies of membrane-free cytoplasmic condensates that form in yeast upon carbon starvation and help protect proteasomes from autophagic degradation. Regulated proteasome subunit homeostasis is thus controlled through cellular probing of the level of proteasome assembly, and the interplay between UPS-mediated degradation or sorting of misfolded proteins into distinct cellular compartments.

Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes.

ALIX/Bro1 proteins are conserved in eukaryotes where they enable targeted trafficking of membrane-associated proteins through the late endosome route to the vacuole. For this, ALIX/Bro1 proteins associate with the endosomal sorting complex required for transport (ESCRT) machinery acting as ubiquitin receptors that recognize and sort protein cargoes by binding to ubiquitin-cargo conjugates. However, recent findings show direct interaction of ALIX and protein cargoes, pointing to the existence of different mechanisms for specific target recognition by ALIX. The catalogue of proteins that interact with the Arabidopsis homologue of ALIX is increasing, including both protein cargoes and regulatory proteins that mediate or modulate ALIX function. In this context, we describe a toolkit of techniques to analyze the effect of ALIX function in the endosomal trafficking of specific cargoes, which could be easily extended to other components of the plant ESCRT machinery.

Qualitative difference of mitochondrial subproteoms of brain RPN10- and RPN13-binding proteins

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. These subunits make basically the same contribution to the subsequent translocation of target proteins to the core part of the proteasome. A comparative study of mouse brain mitochondrial subproteomes bound to Rpn10 and Rpn13 subunits revealed a high specificity of the repertoire of Rpn10 and Rpn13-binding proteins. Moreover, proteins, for which mitochondrial localization or association with mitochondrial membranes was previously shown, prevailed in the case of using the Rpn13 subunit as an affinity ligand (Rpn13-binding proteins). This suggests that Rpn10 and Rpn13 play different roles in the degradation of mitochondrial proteins and MAM.

The Ubiquitin-Binding Protein OsDSK2a Mediates Seedling Growth and Salt Responses by Regulating Gibberellin Metabolism in Rice.

UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins are ubiquitin receptors and transporters in the ubiquitin-proteasome system that play key roles in plant growth and development. High salinity restricts plant growth by disrupting cellular metabolism, but whether UBL-UBA proteins are involved in this process is unclear. Here, we demonstrate that the UBL-UBA protein OsDSK2a (DOMINANT SUPPRESSOR of KAR2) mediates seedling growth and salt responses in rice (Oryza sativa). Through analysis of osdsk2a, a mutant with retarded seedling growth, as well as in vitro and in vivo assays, we demonstrate that OsDSK2a combines with polyubiquitin chains and interacts with the gibberellin (GA)-deactivating enzyme ELONGATED UPPERMOST INTERNODE (EUI), resulting in its degradation through the ubiquitin-proteasome system. Bioactive GA levels were reduced, and plant growth was retarded in the osdsk2a mutant. By contrast, eui mutants displayed increased seedling growth and bioactive GA levels. OsDSK2a levels decreased in plants under salt stress. Moreover, EUI accumulated under salt stress more rapidly in osdsk2a than in wild-type plants. Thus, OsDSK2a and EUI play opposite roles in regulating plant growth under salt stress by affecting GA metabolism. Under salt stress, OsDSK2a levels decrease, thereby increasing EUI accumulation, which promotes GA metabolism and reduces plant growth.

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.

Molecular networks regulating cell division during Arabidopsis leaf growth.

Leaves are the primary organs for photosynthesis, and as such have a pivotal role for plant growth and development. Leaf development is a multifactorial and dynamic process involving many genes that regulate size, shape, and differentiation. The processes that mainly drive leaf development are cell proliferation and cell expansion, and numerous genes have been identified that, when ectopically expressed or down-regulated, increase cell number and/or cell size during leaf growth. Many of the genes regulating cell proliferation are functionally interconnected and can be grouped into regulatory modules. Here, we review our current understanding of six important gene regulatory modules affecting cell proliferation during Arabidopsis leaf growth: ubiquitin receptor DA1-ENHANCER OF DA1 (EOD1), GROWTH REGULATING FACTOR (GRF)-GRF-INTERACTING FACTOR (GIF), SWITCH/SUCROSE NON-FERMENTING (SWI/SNF), gibberellin (GA)-DELLA, KLU, and PEAPOD (PPD). Furthermore, we discuss how post-mitotic cell expansion and these six modules regulating cell proliferation make up the final leaf size.

Preclinical Validation of Ubiquitin Receptor PSMD4 As Therapeutic Target in Multiple Myeloma

Background and Rationale Our preclinical studies have focused on Identification and validation of components within the ubiquitin proteasome system which can be targeted with novel therapies to overcome proteasome-inhibitor (PI)-resistance in multiple myeloma (MM). Our siRNA screening studies identified ubiquitin Receptor (UbR) PSMD4/Rpn10 as a mediator of MM cell growth and survival. Rpn10 is localized on the 19S regulatory lid of the proteasome, and plays a key role in chaperoning ubiquitinated substrate proteins for downstream 20S proteasomal degradation. Here, we show that inhibition of Rpn10 triggers potent anti-MM activity using both in in vitro and in vivo models of MM, including against PI-resistant MM cells. Materials and Methods Rpn10 knockout 293 cell line was generated using CRISPR/Cas9. Rpn10-inducible knockdown (KD) MM.1S cell line was generated using shRNA. Drug sensitivity, cell viability, and apoptosis assays were performed using WST/CellTiter-Glo assay, and Annexin V staining, respectively. Cell signaling and caspase activity were determined by western blotting. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2012, 22:345-358). A xenograft human MM model was used to characterize the role of Rpn10 on tumor progression. Statistical significance was assessed with Student's t test. Results 1) Analysis of MM patient gene expression profiling database showed that Rpn10 expression inversely correlates with overall survival (n=175; p= 0.00064). 2)Real-time PCR, immunoblotting, and immunohistochemistry of MM patient bone marrow showed higher Rpn10 levels in patient MM cells and MM cell lines versus normal cells. 3)Transient transfection of MM cells with Rpn10-siRNA decreased their viability; conversely, transfection with Rpn10-WT specifically rescued cells from growth-inhibitory activity of Rpn10-siRNA. Immunoblot analysis confirmed significant knockdown of Rpn10 by Rpn10-siRNA versus scrambled (scr)-siRNA, and restoration of Rpn10 levels in cells transfected with Rpn10-WT versus Rpn10-siRNA. 4)Genetic blockade of Rpn10 decreased viability in bortezomib-resistant MM cells. 5)CRISPR/Cas9-mediated knockout (KO) of Rpn10 decreased cell growth. 5)Both Rpn10-KO and inducible Rpn10-KD cells showed elevated levels of polyubiquitylated proteins. 6)Inhibition of Rpn10 induced apoptosis, cell-cycle arrest, activation of caspases, and endoplasmic reticulum stress response signaling. 7)Reduced tumor growth was noted in mice xenografted with Rpn10-KD MM.1S cells versus mice engrafted with WT-MM.1S cells. Conclusion Our data demonstrate the therapeutic potential of targeting ubiquitin receptor Rpn10/PSMD4, and provide the preclinical basis for development of Rpn10 inhibitors to overcome PI-resistance in MM. Disclosures Chauhan: C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Proteomic Analysis Identifies Mechanism(s) of Overcoming Bortezomib-Resistance Via Targeting Ubiquitin Receptor Rpn13

Introduction Our studies have focused on delineating ubiquitin proteasome pathway signaling with a goal to identify and validate novel therapeutic targets to overcome proteasome-inhibitor-resistance in multiple myeloma (MM). Our siRNA screening studies showed ubiquitin Receptor (UbR) Rpn13 as a mediator of MM cell growth. Rpn13 is localized on the 19S proteasome, and chaperone ubiquitinated proteins for downstream proteasomal degradation. We showed that blockade of Rpn13 trigger cytotoxicity in MM cells and overcomes bortezomib-resistance (Song Yan et al., Leukemia 2016, 30(9):1877-1886). However, the mechanism(s) whereby Rpn13-inhibition overcomes bortezomib-resistance remains undefined. Here, MM cells were treated with the Rpn13 inhibitor RA190 to identify proteomic alterations and specific downstream substrate proteins/signaling mechanism involved in overcoming bortezomib-resistance. We identified and validated the Copper/Zinc-superoxide dismutase (SOD1), implicated in the regulation of mitochondrial unfolded protein response, as a novel therapeutic target in MM. Materials and Methods MM.1S cells were treated with DMSO or RA190 (250 nM) for 24h and subjected to multiplexed proteomics analysis using tandem mass spectrometry. Data were analyzed using uniprot composite database and SEQUEST-based software platform. MM.1S cells were transiently transfected with scr-siRNA, SOD1-siRNA, control (pCDNA3.1)- or SOD1 (WT)-plasmid using the Nucleofector Kit V. Rpn13 knockout cells were generated using CRISPR/Cas9. Cell viability was assessed using WST-1, and Cell Titer-Glo assay. Alterations in ROS, SOD1, superoxide levels and Proteasome activity were measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Rpn13 inhibitor RA190 and Cu-ZnSOD1 inhibitor LCS-1 were purchased from Calbiochem and Sigma-Aldrich, respectively. Statistical significance was determined using a Student's t test. Results 1) Rpn13 inhibitor RA190 decreases viability in bortezomib-resistant MM cells. 2) Proteomic analysis showed a marked decrease in superoxide dismutase (SOD1) levels (69% ± 2.1%) in RA190- vs DMSO-treated MM cells. No significant decrease in SOD1 expression was noted in bortezomib-treated cells. 3) Transfection with SOD1-WT rescued cells from growth-inhibitory activity of RA190, suggesting that SOD1 mediates Rpn13-inhibition triggered cell death. 4) Both patient MM cells and MM cell lines exhibit higher SOD1 protein levels vs normal plasma cells/PBMCs; 5) SOD1 levels are higher in bortezomib-resistant vs -sensitive MM cells. 6) Gene expression (GEP) analysis showed inverse correlation between SOD1 and overall patient survival (n=175) (p = 0.00054). 7) Direct targeting of SOD1 using siRNA showed significant decrease in viability of MM cell lines, including bortezomib-resistant MM cells. 8) As for SOD1-siRNA, treatment of MM cell lines and primary patient cells with the SOD1 inhibitor LCS-1 decreased their viability (p < 0.05; n=3) without affecting normal PBMCs. 9) LCS-1 inhibits the proliferation of MM cells even in the presence of tumor-promoting BMSCs and plasmacytoid dendritic cells. 10) Mechanistic studies show that similar to RA190, LCS-1-induced MM cell death is associated with: (i) inhibition of SOD1 enzymatic activity and increase in superoxide and reactive oxygen species (ROS) levels; (ii) activation of apoptotic signaling via caspases, p53/p21 axis, and cyto-c release; (iii) decrease in MCL-1, BCL2, CDC2, CDC25C, and cyclin B1; (iv) induction of ER stress response; and (v) accumulation of ubiquitinated proteins and proteasome disassembly. 11) ROS inhibitor N-acetyl-L-cysteine blocked LCS-1-induced cell death and associated signaling. 12) LCS-1, but not RA190, induces cytotoxicity in CRISPR/Cas9-Rpn13-KO cells, confirming our proteomic data showing SOD1 as a downstream target of Rpn13. Conclusion Our proteomic and functional validation studies show that targeting UbR Rpn13 with RA190 overcomes bortezomib-resistance via decreasing cellular SOD1 levels, which therefore represents a target for novel therapeutics to improve patient outcome. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

THE ENDOPLASMIC RETICULUM PROTEIN QUALITY CONTROL ADAPTATION IN A LONG-LIVED C. ELEGANS PROTEASOMAL MUTANT

Protein degradation mechanisms are integral to protein homeostasis. Their reduced efficiency during aging leads to accumulation of misfolded and aggregated proteins which potentiate proteotoxic disorders. Paradoxically, our lab reported that the Caenorhabditis elegans rpn-10(ok1865) proteasome mutant possesses enhanced proteostasis and extended lifespan. RPN-10/PSMD4 is a ubiquitin receptor of the 26S proteasome that targets polyubiquitinated substrates to its catalytic core for degradation. Proteasome dysfunction of the rpn-10 mutant is characterized by reduced, not inhibited, ubiquitin fusion degradation. We ascertained that upregulated autophagy and SKN-1/Nrf-mediated responses partially contribute to the robust rpn-10 mutant phenotype. Further investigation of its underlying mechanism revealed that several ERQC genes are transcriptionally upregulated in the rpn-10 mutant. Thus, we hypothesized that the rpn-10 mutant exhibits improved ER proteostasis which mediates its elevated cellular stress resistance. Accordingly, the rpn-10 mutant shows increased ER stress resistance and altered ER homeostasis. Complementarily, attenuated expression of the aggregation-prone α-1 antitrypsin (ATZ) reporter proves that ER proteostasis is ameliorated in the rpn-10 mutant. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we identified novel player H04D03.3, which is a homolog of the proteasome adaptor ECM29. This suggests that assembly of the rpn-10 mutant proteasome itself critically regulates its ER proteostasis. Moreover, we observed that cytosolic proteostasis and longevity depend on ER master chaperone hsp-3/-4(BiP) and ER ATPase cdc-48.2(p97/VCP), further highlighting ERQC significance in the rpn-10 mutant. Altogether, it appears that mild proteasomal dysfunction induces ERQC adaptation that underlies proteostasis and longevity benefits of the rpn-10 mutant.

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.

The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway.

BackgroundAlthough bone morphogenetic protein 6 (BMP6) signaling pathway has been implicated in many types of cancer, its role of tumorigenesis seems to be controversial and its ubiquitin-modifying mechanisms have not been fully addressed. Our study was designed to investigate how BMP6 signaling pathway is regulated by ubiquitin-modifying systems and to address molecular and clinical significance in colorectal cancers.MethodsHuman deubiquitnase (DUB) siRNA library was used to screen the specific DUB, named PSMD14, involved in BMP6 signaling pathway. Immunoblot, immunoprecipitation and ubiquitination assays were used to analyze targets of the PSMD14. A role of PSMD14-mediated BMP6 signaling pathway for malignant cancer progression was investigated using in vitro and in vivo model of colorectal cancers as well as clinical samples of colorectal cancer patients.FindingsThe deubiquitinase PSMD14 acts as a positive regulator for the initiation of the BMP6 signaling pathway through deubiquitinating K48-linked ALK2 type I receptor ubiquitination mediated by Smurf1 E3 ligase, resulting in increased stability of the ALK2. This role of PSMD14 is independent of its intrinsic role in the 26S proteasome system. Furthermore, either PSMD14 or ALK2 depletion significantly decreases tumorigenesis of HCT116 colorectal cancer cells in a xenograft model as well as cancer stemness/chemoresistance, and expression of the PSMD14 and ALK2 gene are correlated with malignant progression and the survival of colorectal cancer patients.InterpretationThese findings suggest that the PSMD14-ALK2 axis plays an essential role in initiation of the BMP6 signaling pathway and contributes to tumorigenesis and chemoresistance of colorectal cancers.

Discovery of Interacting Proteins of ABA Receptor PYL5 via Covalent Chemical Capture.

Abscisic acid (ABA) is a key phytohormone with diverse functions in plants, and its signal transduction is mainly mediated by ABA receptors termed PYR/PYL/RCARs (hereafter referred to as PYLs) through the PYLs-PP2Cs-SnRK2s regulatory systems. However, the model failed to account for the roles of some important known regulators of ABA physiology. Given the central role of PYLs in ABA signal transduction, we therefore speculated that ABA receptors PYLs might be involved in regulatory pathways other than PP2Cs. Thus, a comprehensive analysis of PYLs-interacting partners could greatly facilitate the identification of unknown regulatory pathways, advancing our knowledge of the ABA signaling mechanism. Herein, we present a strategy involving covalent chemical capture coupled with HPLC-MS/MS analysis, to profile PYL5-interacting partners in plant cell lysates. With this strategy, three new PYL5-interacting partners, ubiquitin receptor RAD23C, COP9 signalosome complex subunit 1 (CSN1), and cyclase-associated protein 1 (CAP1), along with their key binding sites with PYL5 were identified. Among these proteins, CAP1 was verified to interact with PYL5 both in vitro and in vivo. The discovery of a new PYL5 binding partner showed the versatility of covalent chemical cross-linking and laid the foundation for future efforts to further elucidate the ABA signaling mechanism.

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.

A Small Molecule-induced Targeted Degradation of Ubiquitin Receptor Rpn13 as a Novel Therapeutic Strategy in Multiple Myeloma

Proteasome inhibitors (PIs) represent major advance in the treatment of multiple myeloma (MM); however, PI-therapy is associated with adverse effects and the development of drug-resistance. Strategies to overcome PI-resistance include targeting ubiquitin proteasome system (UPS) upstream of 20S proteasome. Our earlier studies identified one such target ubiquitin Receptor (UbR) Rpn13 as a mediator of MM cell growth and survival. Rpn13 is associated with 19S regulatory lid of proteasome and play a key role in recognizing ubiquitylated proteins and directing their degradation via downstream 20S proteasome.

Inhibition of Ubiquitin Receptor PSMD4/Rpn10 as Therapeutic Strategy to Overcomes Bortezomib-Resistance in Multiple Myeloma cells

and Rationale Proteasome inhibitors (PIs) represent major advance in the treatment of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, PI-therapy is associated with adverse effects and the development of drug-resistance. Strategies to overcome PI-resistance include targeting ubiquitin proteasome system (UPS) upstream of 20S proteasome subunit. One such exemplary target is ubiquitin receptor PSMD4/Rpn10, which is localized on 19S regulatory lid of proteasome, and play a key role in recognizing ubiquitylated substrate proteins for degradation via downstream 20S proteasome.