Tandem Ubiquitin Binding Entities Research Articles

Central Angiotensin II regulates Protein Inhibitor of Neuronal Nitric Oxide Synthase through post‐translational mechanisms in the Paraventricular Nucleus resulting in increased Sympathetic outflow

Increased activation of Renin‐Angiotensin System in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in hypertension and chronic heart failure (CHF). Previously we have shown increased protein expression of the PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. To elucidate the molecular mechanism of increased PIN expression post‐transcriptionally, we used Sprague‐Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14 days, 0.5 μl/h) through osmotic mini‐pumps and NG108‐15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg) and baseline renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max activity). Ang II infusion increased the expression of the PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) a with concomitant decrease in PIN‐Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, PIN translation was inhibited in vitro using protein synthesis inhibitor cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. Ang II‐mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II‐mediated post‐translational stabilization of PIN. Ubiquitination assay in cells transfected with pCMV‐(HA‐Ub) 8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). These results suggest that PIN is targeted for rapid degradation by the ubiquitin‐proteasome and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN. It of interest to note that previously we observed that there was a decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (0.63 ± 0.05* CHF vs. 1.00 ± 0.10 Sham). Taken together, our studies suggest that increased central levels of Ang II contribute to the upregulation of PIN expression in the PVN via decreasing the ubiquitination which may be involved in reducing the expression of dimeric nNOS of pre‐autonomic neurons in the PVN resulting in the increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 & HL62222.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A038: Establishing ubiquitylation pattern in tissues and cells efficient method to monitor cancer drug function and biomarkers

Abstract The cell regulates its function by altering ubiquitylation status of many key proteins, especially oncoproteins. As a result, these ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anticancer targets. However, to date, the only effective way to measure these effects has been by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, which are either insensitive, low throughput, or highly intensive. Here, we describe two Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiQuant S and UbiTest, that provide a platform to efficiently detect ubiquitylation of anticancer targets. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in a cell or tissue. Ubiquitylated proteins and the nature poly-ubiquitin are identified by energy transfer between tagged TUBE and antibody against the target protein that is poly-ubiquitylated. Interaction between TUBE and antibody is registered in a label-free manner in a solution. In case the level of ubiquitylated protein is extremely low or cannot be detected by antibody, ubiquitylated proteins can be affinity purified by TUBEs attached to agarose or magnetic beads and FRET or alphaLISA experiment is repeated. Furthermore, we have extended the technology to identify K63- and K48-linked poly-ubiquitin proteins by poly-ubiquitin chain selective TUBEs. UbiTest is an immunoblot-based assay, which can be used to verify the result of UbiQuant S. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. Several important therapeutic targets have been used to verify these assays, including Cbl-b and USP7 immune oncology drug targets. The high efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples from DUBs and ubiquitin ligase inhibitors will illustrate the power of this system. These novel technologies will equip researchers with versatile tools to better understand the role of ubiquitylation in cancer treatment and monitoring biomarkers in response to drug action. Citation Format: Xiaolong Lu, Rajesh Singh, Chengcheng Song, Peter Foote. Establishing ubiquitylation pattern in tissues and cells efficient method to monitor cancer drug function and biomarkers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A038.

Abstract B074: Ubiquitin proteomics: profiling the landscape of ubiquitin modification by ubisite-omics

Abstract The ubiquitin pathway has emerged as a promising druggable target for cancer. Ubiquitylation regulates various cellular functions including cell cycle, differentiation, apoptosis, and stress response. Ubiquitylation of proteins is encrypted bya variety of chain-selective K48, K63, K6, K11 poly-ubiquitins as well as mono-ubiquitin attached to target proteins. Proteomics provides an effective approach for decoding the complex ubiquitin signals. Ubiquitylation is a very dynamic process and ubiquitylated proteins are low in abundance and stoichiometry. Enrichment of ubiquitylated proteins by affinity metrics TUBE (Tandem Ubiquitin Binding Entities) is essential to improve quantitative, reproducible, and sensitive proteomics analysis. The most common approach is to use antibody that pulls down DiGlyGly-lysine (C-terminal di-Gly of ubiquitin attached to epsilon amino group of lysine), the so-called “ubiquitin remnant.” However, ubiquitylation from ubiquitin-like proteins (UBLs) such as SUMO, NEDD8, and ISG15 that are undistinguishable by DiGlyGly-lysine antibody makes it impossible to assign a role for ubiquitin or UBLs. Here we demonstrate a novel ubiquitylation-specific proteomics approach. This method utilizes UbiSite antibody that recognizes the ubiquitin C-terminus (13 a.a.), which creates the specificity for ubiquitin remnant. HepG2 and Jurkat cells were chosen as model systems. The alteration in cellular protein ubiquitylation were promoted by treatment with the inhibitor of a proteasome-associated DUB, USP14, a USP7 DUB inhibitor that is an immune oncology target or bortezomib, a 26S proteasome inhibitor. Cell lysates were digested with Lys C and immuneprecipitated with UbiSite antibody. Enriched ubiquitylated peptides were further digested with trypsin and analyzed by LC-MS/MS. The total proteome and ubiquitome were compared among treated and untreated samples. In addition, the ubiquitome of proteins, the site-specific ubiquitylation levels, were also compared with other published studies. We have identified a number of novel ubiquitylated proteins using the UbiSite proteomics approach. We believe this approach will facilitate discovery of pharmacologic markers for drugs, uncover new substrates for DUBs and ubiquitin ligases, and open doors for cell biologists to specifically identify genome-wide patterns of poly-ubiquitylated proteins. Citation Format: Chengcheng Song, Vyacheslav Akimov, Peter Foote, Xiaolong Lu, Blagoy Blagoev, Rajesh Singh. Ubiquitin proteomics: profiling the landscape of ubiquitin modification by ubisite-omics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B074.

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo.

Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods. Specifically, we employ tandem ubiquitin binding entities (TUBEs) and the K29-selective Npl4 Zinc Finger 1 (NZF1) domain from the deubiquitinase TRABID to enrich for chains from human cells. Minimal trypsinolysis followed by high resolution mass spectrometric analysis reveals that Ub chain branching can indeed be detected using both Ub binding domains (UBDs) tested at endogenous levels. We find that ∼1% of chains isolated with TUBEs contain Ub branch points, with this value rising to ∼4% after proteasome inhibition. Electron-transfer dissociation (ETD) analysis indicates the presence of K48 in these branched chains. The use of the NZF1 domain reveals that ∼4% of the isolated chains contain branch points with no apparent dependence on proteasome inhibition. Our results demonstrate an effective strategy for detecting and characterizing the dynamics of branched conjugates under different cellular conditions.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Biological significanceIn this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored.

Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs.

Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.

Method to Measure Ubiquitination of NLRs.

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here, we describe a method to detect polyubiquitination of NRLP3 using two different approaches: (1) detection with an ubiquin antibody or (2) using TUBE (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLR or other components of the inflammasome.

Mass spectrometry analysis of K63-ubiquitinated targets in response to oxidative stress

The data described here provide the first large-scale analysis of lysine 63 (K63)-linked polyubiquitin targets. Protein ubiquitination is a prominent post-translational modification, and a variety of ubiquitin chains exists, serving a multitude of functions [1]. The chains differ by the lysine residue by which the ubiquitin monomers are linked. We used yeast Saccharomyces cerevisiae subjected to oxidative stress as a model to study K63 ubiquitination. K63 ubiquitinated targets were pulled-down by the K63-TUBE system (Tandem Ubiquitin Binding Entities) and analyzed by SILAC-based mass spectrometry [2]. The data are associated to the research article ‘K63 polyubiquitination is a new modulator of the oxidative stress response’ [3]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000960.

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

BackgroundThe ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery.MethodsPlasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors.ResultsIn both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition.ConclusionsThe two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

Nedd4 family interacting proteins activate ubiquitylation pathways to limit ERK signaling following TCR engagement (IRM7P.702)

Abstract Ubiquitylation tunes signaling pathways in stimulated T cells to regulate activation and function. We have found that, in vitro, Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 activate Nedd4-family catalytic E3 ligases with known roles in T cells. Ndfip1 negatively regulates T cell activation and Th2 polarization, but the in vivo role of Ndfip2 is unknown. We generated Ndfip2-/- mice and found that Ndfip2 is not a prominent negative regulator of T cell activation or Th2 polarization. However, loss of Ndfip2 exacerbates the inflammatory Ndfip1-/- phenotype, suggesting that Ndfip2 dampens inflammation. We generated mixed fetal liver chimeras and found that Ndfip1/Ndfip2 deficiency leads to T cell intrinsic hyperactivity. To identify signaling pathways affected by loss of Ndfips, we used semiquantitative proteomics to compare stimulated control and knockout CD4+ T cells. Pathway analysis revealed increased MEK1 activity in knockout cells, which was confirmed by observing increased ERK phosphorylation in stimulated Ndfip1/Ndfip2 deficient CD4+ T cells. To identify aberrant ubqiuitylation of proteins upstream of ERK, we have taken a quantitative proteomic approach, using Tandem Ubiquitin Binding Entities (TUBEs) to enrich for polyubiquitylated proteins in isotope labeled control and Ndfip deficient CD4+ T cells. This approach has yielded several potential targets of Ndfip-dependent ubiquitylation in CD4+ T cells that are currently under investigation.

Poly‐ubiquitin Chains Paradox: Development of Novel and Selective Poly‐ubiquitin Binding Domains

Ubiquitylation occurs through linkage between the C-terminus of Ub and the ε-amino group of a Lys on the target substrate. Ub has 7 Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in generating poly-Ub chains. The ability of Ub to form polymers is central to the versatility of this system in regulating cellular processes. The most extensively characterized of these polymers are linked through either K48 or K63. K48-linked polyUb predominantly targets proteins for proteasomal degradation, whereas K63-linked polyUb appears to regulate protein function, localization, or interactions. K11-linked polyUb has been implicated in mitotic regulation and ERAD. It is apparent that different poly-ubiquitin linkages convey different information to the cell and strongly suggests that the cell contains elements capable of decoding this information, i.e. linkage specific ubiquitin binding domains. Development of Poly-Ubiquitin selective tools will revolutionize the field of ubiquitin. Most UBDs described to date show little ability to discriminate between different linkages; although, it is possible to construct linkage specific tandem ubiquitin binding entities (TUBEs) by manipulating the spacing and rigidity of the linkers between tandem UBDs. We and our collaborators have constructed a series of K63-specific UBDs that are useful for Far Western blotting and “pull-down” type experiments for evaluating the levels and identities of proteins bearing K63-linked polyubiquitin chains. We report here on the development of new TUBE-type reagents that distinquish between chain linkages and can be used in dissecting the exact role of the different linkages in ubiquitin biology. This information will impact on our understanding of the UPP and suggest new targets for intervention.

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.

Analysis of PTEN ubiquitylation and SUMOylation using molecular traps

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono- or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination

ObjectivesTo determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA).MethodsBovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human...

Poly‐ubiquitin chains paradox: development of novel and selective poly‐ubiquitin binding domains (LB184)

Ubiquitylation occurs through isopeptide linkage between the C‐terminus of Ub and the ε‐amino group of a lysine residue on the target substrate. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can participate in further ubiquitylation, generating poly‐Ub chains. The ability of Ub to form polymers through various lysines as well as the NH2‐terminus appears to be central to the versatility of this system in regulating cellular processes. The most extensively characterized of these polymers are linked through either K48 or K63. K48‐linked polyUb predominantly targets proteins for proteasomal degradation, whereas K63‐linked polyUb appears to regulate protein function, subcellular localization, or protein‐protein interactions. A growing body of evidence now implicates K11‐linked polyUb in mitotic regulation and endoplasmic reticulum associated degradation (ERAD). It is apparent from the above, that different poly‐ubiquitin linkages convey different information to the cell and suggests strongly that the cell contains elements capable of decoding this information, i.e. linkage specific ubiquitin binding domains. Development of Poly‐Ubiquitin selective tools will revolutionize the field of ubiquitin. Most UBDs described to date show little ability to discriminate between different linkages; although, it is possible to construct linkage specific tandem ubiquitin binding entities (TUBEs) by manipulating the spacing and rigidity of the linkers between tandem UBDs. We and our collaborators have constructed a series of K63‐specific UBDs and demonstrated their utility for Far Western blotting and “pull‐down” type experiments for evaluating the levels and identities of proteins bearing K63‐linked polyubiquitin chains under a variety of treatments. We will describe the results of proteomic experiments in which we identified new classes of UBDs. When validated, this information will have a major impact on our understanding of the functioning of the Ubiquitin‐Proteasome Pathway and suggest new targets for intervention.Grant Funding Source: Supported by R43CA165561

Lenalidomide Induces A Ribosomal Stress Response In Multiple Myeloma (MM) Cells

BackgroundDeregulation of ribosome biogenesis is associated with carcinogenesis and in MM two of the most common recurrent mutations (DIS3 and FAM46C) are implicated in ribosomal decay and translational control. Beside its role in the regulation of protein synthesis, the importance of ribosome biogenesis is underscored by the observation that the impairment of this process leads to a ribosomal stress response with induction of p53, inhibition of c-Myc and cell cycle arrest. These effects are mediated by the direct binding of ribosomal proteins (RPs), particularly RPL11 and RPL5 to Mdm2 and c-Myc. Immunomodulatory drugs (IMiDs) anti-MM effects require their binding to Cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex with an ensuing down-regulation of c-Myc and up-regulation of p21. In the present study, we delineated the mechanisms through which IMiDs mediate these effects in MM cells. Methods and ResultsIn order to identify ubiquitylated substrates that are modified by lenalidomide (Len) treatment, we performed a ubiquitin-proteome pull-down using Tandem Ubiquitin Binding Entity (Lifesensors) coupled with quantitative mass-spectroscopy proteomics (iTRAQ) in OPM2 cells exposed to Len (10 μM). Several ribosomal proteins (RPs) - S25, S26, S20 & S28 - were increased in Len treated samples suggesting that the Cul4a-CRBN complex may regulate RPs stability and hence IMiDs binding to cereblon may trigger a ribosomal stress response. Immunoblot analysis of MM cells exposed to Len lead to a rapid decrease in c-Myc expression (within 30 min) that significantly preceded the downregulation of IRF4 consistent with an IRF4-independent mechanism for c-Myc down-regulation. Furthermore, Len treatment transiently stabilised and subsequently down-regulated MDM2 expression with up-regulation of p53 and its down-stream targets (p21, PUMA). Under ribosomal stress conditions, polysome-free RPs are released into the nucleoplasm where they bind Mdm2 and suppress its E3 ligase activity. Consistent with this effect, in Len treated cells MDM2 co-immunoprecipitated with RPL11 and RPL5 with p53 protein stabilization (no change in p53 mRNA). In addition, an increase in the interaction between RPL11 and c-Myc was also observed consistent with the reported role for RPL11 in the post-transcriptional regulation and suppression of c-Myc. Furthermore, treatment with Lenalidomide supressed ribosomal RNA (rRNA) transcription with inhibition of pre-rRNA (47S) processing. The examination of polysome fractions (sucrose gradients) in Len treated MM cells (OPM2 and MM1S) revealed a striking reduction of 60S and 80S as well as polysomes fractions, an effect similar to that Actinomycin D (5 nM), a known RNA PolI inhibitor and potent ribosome stress inducer. Furthermore Len treatment significant reduced RPL11 within the 60S and 80S ribosome fractions. Of note, CRBN knockdown abrogated all these effects suggesting that Len binding to CRBN is upstream of this ribosome stress response. In order to investigate whether the down-regulation of c-Myc is the primary event leading to the disruption of ribogenesis, we transiently and stably silenced RPL11 in MM cells. RPL11 silencing nearly fully protected MM cells from the effects of Len and importantly it completely reversed Len-induced Mdm2 E3 ligase inhibition with up-regulation of p53, p21 and suppression of c-Myc and IRF4. ConclusionTaken together our data indicate that treatment with lenalidomide suppresses ribogenesis and induces a ribosomal stress response downstream of Cul4a-CRBN but upstream of c-Myc suppression and p53 induction. These effects likely result from the IMiDs-induced modification of the Cul4a-CRBN ubiquitome and the regulation of RPs integration into ribosomal subunits. These findings also explain the observed clinical activity of IMiDs in other ribosomopathies like 5q- MDS and Diamond-Blackfan anemia Disclosures:No relevant conflicts of interest to declare.

Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing tandem ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.

Lenalidomide Suppresses 5′-Cap-Independent C-MYC Translation in Multiple Myeloma Cells

Abstract 3943 Background:Immunomodulatory drugs (IMiDs) are highly active in the treatment of multiple myeloma (MM). Blockade of the Wnt-GSK3β-beta catenin axis and suppression of eIF4E-C/EBPβ-dependent IRF4 expression were previously shown to mediate some of the IMiDs cytotoxic effects. In particular, cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex, was recently identified as an essential requirement for IMiDs anti-MM activity. In the present study, we examined how lenalidomide treatment modifies MM cells ubiquitome downstream of the Cul4A-DDB1-CRBN E3 ligase complex with the intent to identify novel molecular targets mediating IMiDs anti-MM effects. Methods and Results:Ubiquitin-proteome pull-down using Tandem Ubiquitin Binding Entity (TUBE, Lifesensors) coupled with quantitative mass-spectroscopy based proteomics (iTRAQ) was performed to identify modification to OPM2 cells ubiquitin-proteome induced by lenalidomide (10 μM for 24 hours) versus vehicle control treatment. Among the differentially modified ubiquitilated targets, histone family members H4, H2A.Z, H2B (known substrates of the Cullin-Ring Ub E3 ligases) and the 40S ribosomal proteins RPS25 and RPS26 were more than 2 fold increased in lenalidomide treated samples. Ribosomal protein RPS25 is required for internal ribosomal entry site (IRES) and 5′-cap-independent proteosynthesis. To examine the function of RPS25 in MM, siRNA-KD experiments were carried out in 2 human MM cell lines (OPM2 and MM1S). RPS25 silencing significantly (30–40%) reduced MM cells viability (MTT assay) when compared to non-targeting siRNA controls. Of interest, MM cells surviving RPS25 silencing were more resistant to IMIDs with approximately 20 to 30% reduction in lenalidomide induced cells death (Annexin V staining and MTT assay). Since, the 5′ UTR of c-myc is known to contain an IRES segement and c-myc translation can therefore be initiated by internal ribosome entry (RPS25 mediated) as well as by cap-dependent mechanisms, we next examined whether lenalidomide may downregulate c-myc through an IRF4-independent and rather RPS25-IRES dependent mechanism. Following 4 hours exposure to lenalidomide, C-MYC protein was significantly downregulated (western blot analysis) in OPM2 and MM1S cells, with no changes to IRF4 (protein or mRNA) or c-myc mRNA (qRT-PCR) at this early time point. No changes were observed in IRF4 until 24–48 hours of treatment with lenalidomide, findings consistent with an IRF4-independent C-MYC downregulation. Using the bicistronic (pRMF) luciferase reporter plasmid in which the upstream cistron encodes a Renilla luciferase translated in a cap-dependent manner, and the downstream cistron encoding a firefly luciferase translated in a cap-independent manner because of c-myc IRES insertion between the two cistrons, we examined whether treatment with lenalidomide modifies c-myc IRES ribosomal entry and translation. Following lenalidomide treatment, MM1S-pRMF transfected cells had a significant reduction in c-myc cap-independent translation as indicated by the reduction in the firely/renilla luminescence ratio. These findings were further supported by the use of the m-TOR inhibitor Rapamycin, an inhibitor of cap-dependent protein translation. Co-treatment of MM1S and OPM2 cells for 4 hours with the combination of Rapamycin (10 nM) and lenalidomide (10 μM) resulted in further reduction in C-MYC protein levels compared to either drugs alone. These results are consistent with an independent and additive (5′-cap-dependent with Rapamycin and independent with lenalidomide) rather than epistatic (both 5′-cap dependent) effects on c-myc translation. Conclusions:Taken together our data indicate that treatment with lenalidomide suppresses c-myc translation in an IRF4/5′-cap-translation independent mechanism (early time point), an effect possibly resulting from a Cul4a-CRBN regulation of RPS25, a key protein-mediating ribosomal entry. Disclosures:Neri:Johnson ans Johnson: Research Funding. Bahlis:Johnson and Johnson: Honoraria, Research Funding; Celgene: Honoraria.

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins

The ubiquitin proteasome pathway controls the cellular degradation of ~80–90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.