Tandem Ubiquitin Binding Entities Research Articles

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 poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins.

Purification and Detection of Ubiquitinated Plant Proteins Using Tandem Ubiquitin Binding Entities.

The timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasome or removal of modifications by deubiquitination enzymes (DUBs). Tandem ubiquitin binding entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBs. Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEs after transient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitination of a target protein. This methodology provides an effective method for identification of ubiquitin ligase substrates and can be coupled with TUBEs targeting specific ubiquitination linkages.

Profiling the ubiquitinated proteome in human cells

Ubiquitination is a post‐translational modification that determines the half‐life of many cellular proteins by conjugating a chain of the small protein ubiquitin to a target protein. The ubiquitin chain is then recognized by the 26S proteasome, and subsequently, the ubiquitinated protein is degraded. This ubiquitin‐proteosome system (UPS) contains various components that function cooperatively to determine the ubiquitination status of cellular proteins that play key roles in biological events. It has become clear that mis‐regulation of protein ubiquitination can lead to diverse human diseases, and multiple UPS components have been identified as promising drug targets. To facilitate studies that define roles of the UPS components in protein ubiquitination, a method that probes the ubiquitination status of the whole proteome in human cells is necessary. Tandem ubiquitin binding entities (TUBEs) is a synthetic protein that binds poly‐ubiquitin chains with a dissociation constant in the nanomolar range. Therefore, TUBEs can recognize the poly‐ubiquitinated protein and enrich them from human cell lysate. Combining the TUBE‐dependent affinity precipitation, stable isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry, we are establishing a method for quantitatively accessing the ubiquitinated proteome in human cells. To preserve the poly‐ubiquitinated protein, HEK293 cells were treated with bortezomib, a proteosome inhibitor, to prevent the ubiquitinated proteins from degradation. To perform the affinity precipitation, the recombinant TUBE was conjugated to biotin and immobilized on resin via the Streptavidin‐biotin interaction. The lysate of the bortezomib‐treated cells was incubated with the immobilized BiotinTUBE to enrich poly‐ubiquitinated proteins, which were then digested by the trypsin protease. Following the trypsin digestion, ubiquitinated proteins generated peptides with the Lys‐ɛ‐Gly‐Gly (K‐ɛ‐GG) remnant, which was further immunoprecipitated by antibodies specifically recognizing the K‐ɛ‐GG remnant and identified by mass spectrometry. To probe proteins with different ubiquitination status under different conditions, SILAC was used to grow one cell sample in normal (light) media and the other cell sample in (heavy) media containing stable isotope labeled amino acids. By comparing the abundance of the light versus heavy peptides from the same ubiquitinated protein, changes in the ubiquitination status of each detected protein can be determined. We anticipate that this method will help profile changes in the ubiquitinated proteome when the UPS components are mutated or interfered by drugs.

Mass Spectrometry‐based Proteomics in Deciphering Hormonal Regulation of Ubiquitin Linkages

Hormones such as insulin and glucagon regulate metabolic organ liver. Ubiquitin (Ub) is a 76‐amino acid protein that can be attached on a lysine of the substrate protein. Ub posttranslational modification (PTM) is unique in its diversity as many possible mono‐ and multiple Ub molecules can be attached to protein substrates leading to homotypic and mixed linkages. The type of linkages added determine the functional outcomes such as protein degradation or localization changes. Hormones regulate protein PTM and metabolic disorders such as diabetes involve dysregulation of hormonal signaling. We hypothesized that hormones regulate Ub linkage profile and dysfunction of this regulation is a part of diabetes pathophysiology. We studied the effect of insulin and glucagon on protein ubiquitination in HepG2 cells. We utilized Tandem Ubiquitin Binding Entities (TUBE) and immunoprecipitation strategies for linkage‐specific ubiquitin protein enrichment and further analyzed them by western blotting and proteomics. Our preliminary data indicates differential regulation by hormones of the protein Ub linkage profile. Significant leads obtained from proteomic studies will be further characterized. In addition, we aim to study the ubiquitin linkage profiles in tissues obtained from rodent diabetes models. Our study has potential to find key targets involved in hormonal action and their dysfunction in diabetes.

The global proteome and ubiquitinome of bacterial and viral co-infected bronchial epithelial cells

Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial-viral co-infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as a line of defense and detect pathogens by a high variety of pattern recognition receptors. The post-translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial-viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens. SignificanceViral and bacterial co-infections of the lower respiratory tract are a burden for health systems worldwide. Therefore, it is necessary to elucidate the complex interplay between the host and the infecting pathogens. Thus, we analyzed the proteome and the ubiquitinome of co-infected bronchial epithelial cells to elaborate a potential synergism of the two infecting organisms. The results presented in this work can be used as a starting point for further analyses.

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation.

Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.

Advances in the application of affinity separation for analyzing protein ubiquitination

蛋白质泛素化是真核生物最普遍、最复杂的翻译后修饰方式之一,在细胞的信号转导、生长、发育、代谢等生命过程中发挥着重要作用。泛素化过程的失调则与神经退行性疾病、炎症反应、癌症等重大疾病的发生发展密切相关。分析和研究蛋白质泛素化的结构与功能,可望为认识生命、探索疾病调控内在规律和发现新的诊断策略提供重要信息。生命体系的高度复杂性,泛素化修饰位点、结构类型的多变和多样性,时空动态变化等特点给蛋白质泛素化分析研究带来了巨大的挑战。亲和分离以其高选择性成为泛素化蛋白质结构与功能研究的有力工具。免疫亲和分离法基于抗原-抗体相互作用,是最为经典的分离分析方法,已广泛应用于泛素化蛋白质或肽段的富集分离。源于天然泛素受体的泛素结合结构域(ubiquitin binding domains, UBDs)可与泛素或多聚泛素链相互作用。UBDs和基于此发展起来的串联泛素结合实体(tandem ubiquitin-binding entities, TUBEs)已成为蛋白质泛素化功能研究的热门识别分子。各种多肽类化合物的发展也为蛋白质泛素化的结构和功能解析提供新工具。此外,多种亲和识别配基的联合使用,在蛋白质泛素化修饰的高特异性、高灵敏度分析中展现了独特的优势,为认识生命体内的泛素化修饰提供了重要保障。该文对亲和分离方法在蛋白质泛素化修饰分析中的应用及进展进行了综述。

A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets

The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.

Abstract MP49: Central Angiotensin II Mediates Neurogenic Hypertension Through Hif1-Alpha/NMDAR Axis In The Paraventricular Nucleus Of The Hypothalamus (PVN)

Central activation of the renin-angiotensin system and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of hypertension. The specific neural substrates and the possible molecular mechanisms for the sympathoexcitation remain unclear. The glutamatergic activation via N-methyl-d-aspartate receptors (NMDAR) in the PVN enhances sympathetic outflow in hypertension. Previously, using Chromatin immunoprecipitation assay (ChiP) we have shown that Hypoxia-Inducible Factor-1 (heterodimer of HIF-1 α and HIF-1 β subunits ) binds to HREs elements in the NMDA-NR1(structural subunit of NMDAR) promoter in nucleus and increases transcription. In this study, central infusion of Ang II (20 ng/min, 14days, 0.5μl/h, i.c.v) through osmotic mini-pumps in Sprague-Dawley rats increased renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max) and mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg). At the same time, there was an increased expression of HIF-1α mRNA (2.79 fold) and HIF-1α protein (1.54 fold), as well as the expression of NMDA-NR1 mRNA (3.17 fold) and NMDA-NR1 protein levels (1.52 fold) in the PVN of Ang II infused group. Direct application of Ang II (1μM) induced an increase in the expression of HIF-1α protein (0.74 ± 0.03* vs. 0.50 ± 0.04) as well as decreased expression of prolyl hydroxylase domain protein 2 (an enzyme hydroxylating the proline residues of HIF-α, inducing subsequent ubiquitination and proteasomal degradation) (1.47 ± 0.14* vs. 0.50 ± 0.03) in NG108-15 neuronal cells. Tandem Ubiquitin-Binding Entities assay showed decreased HIF-α -Ub conjugates in Ang II-treated cells (0.65 ± 0.09* vs. 1.31 ± 0.10) suggesting a role for Ang II in post-translational stabilization of HIF-α. Further, silencing of HIF-1α (~60%) with siRNA in NG108-15 cells leads to a decrease in the expression of NMDA-NR1 induced by Ang II, compared with scrambled siRNA (0.47 ± 0.05* vs 0.80 ± 0.04). Taken together, these studies suggest that elevated central Ang II upregulates the expression of HIF-1α at transcriptional as well as post-translational level, which potentiates glutamatergic tone by enhanced expression of NMDA-NR1 in the PVN leading to heightened glutamatergic tone with a consequent increase in sympathetic outflow resulting in hypertension.

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs).

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension

Activation of renin-angiotensin- system, nitric oxide (NO•) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases, including hypertension. Previously we have shown increased protein expression of 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 heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 μl/h) or saline as vehicle (Veh) for 14 days 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 renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 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. 1.21 ± 0.06 LC) while AT1R blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO• on pre-autonomic neurons in the PVN resulting in increased sympathetic outflow.

Proteomic analysis of the poly‐ubiquitin profiles in Alzheimer's Disease

Several neurodegenerative diseases including Alzheimer's Disease (AD), have characteristic ubiquitin positive pathological protein aggregates. Currently, the specific ubiquitylation protein substrates associated with these aggregates in AD brain is poorly understood. We analyzed control and AD postmortem brain tissues to examine the changes in the proteome and ubiquitylation substrates associated with AD pathology. Affinity capture using tandem ubiquitin binding entities (TUBEs) was utilized to enrich poly‐ubiquitylation substrates from AD and age‐matched control postmortem brain tissues (n=3 each group). Label‐free mass spectrometry (MS) based proteomic analysis identified a total of 2112 unique proteins across all samples and bead controls. After controlling for non‐specific binding, we identified 197 proteins that were at least two‐fold enriched in AD compared to control cases. Some of these targets include ubiquitin, Tau, p62 sequestosome and 14‐3‐3 proteins, which are known to associate with AD neuropathology. A cross comparison with previous proteomic studies revealed that 108 of those 197 proteins have increased site‐specific levels of ubiquitylation in AD brain. Tau poly‐ubiquitylation from AD brain homogenates was confirmed by affinity capture using either pan or linkage‐specific (Lys48 and Lys63) poly‐ubiquitin TUBEs and immunoblotting. These findings show the utility of MS to map ubiquitylated substrates in the human brain and provide a better understanding of the mechanisms underlying post‐translational modifications of pathological proteins in AD.Support or Funding InformationNational Institute on Aging (U01AG046161‐02, R21AG054206, 5R01AG053960, RF1AG057470, and RF1AG057471) and the NINDS Emory Neuroscience Core (P30NS055077).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using proteomics to identify ubiquitin ligase-substrate pairs: how novel methods may unveil therapeutic targets for neurodegenerative diseases.

Ubiquitin ligases play an integral role in fine-tuning signaling cascades necessary for normal cell function. Aberrant regulation of ubiquitin ligases has been implicated in several neurodegenerative diseases, generally, due to mutations within the E3 ligase itself. Several proteomic-based methods have recently emerged to facilitate the rapid identification of ligase-substrate pairs-a previously challenging feat due to the transient nature of ligase-substrate interactions. These novel methods complement standard immunoprecipitations (IPs) and include proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, tandem ubiquitin-binding entities (TUBEs), and a molecular trapping unit known as the NEDDylator. The implementation of these techniques is expected to facilitate the rapid identification of novel substrates of E3 ubiquitin ligases, a process that is likely to enhance our understanding of neurodegenerative diseases and highlight novel therapeutic targets for the treatment of neurodegenerative diseases.

TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes.

Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).

Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE.

Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).

Quantitative Analysis of the Brain Ubiquitylome in Alzheimer's Disease.

Several neurodegenerative diseases including Alzheimer's Disease (AD) are characterized by ubiquitin-positive pathological protein aggregates. Here, an immunoaffinity approach is utilized to enrich ubiquitylated isopeptides after trypsin digestion from five AD and five age-matched control postmortem brain tissues. Label-free MS-based proteomic analysis identifies 4291 unique ubiquitylation sites mapping to 1682 unique proteins. Differential enrichment analysis shows that over 800 ubiquitylation sites are significantly altered between AD and control cases. Of these, ≈80% are increased in AD, including seven poly ubiquitin linkages, which is consistent with proteolytic stress and high burden of ubiquitylated pathological aggregates in AD. The microtubule associated protein Tau, the core component of neurofibrillary tangles, has the highest number of increased sites of ubiquitylation per any protein in AD. Tau poly ubiquitylation from AD brain homogenates is confirmed by reciprocal co-immunoprecipitation and by affinity capture using tandem ubiquitin binding entities. Co-modified peptides, with both ubiquitylation and phosphorylation sites, are also enriched in AD. Notably, many of the co-modified peptides mapped to Tau within KXGS motifs in the microtubule binding region suggesting that crosstalk between phosphorylation and ubiquitylation occurs on Tau in AD. Overall, these findings highlight the utility of MS to map ubiquitylated substrates in human brain and provides insight into mechanisms underlying pathological protein posttranslational modification in AD.

Abstract 106: Central Angiotensin II Mediates Neurogenic Hypertension Through Post-Translational Regulation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of the Hypothalamus

Activation of renin-angiotensin- system, nitric oxide (NO) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases including hypertension. Previously we have shown Ang II enhances PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) mediated ubiquitination of nNOS in the PVN of rats with heart failure. To further elucidate the mechanism by which Ang II increases sympathetic outflow by regulating PIN mediated nNOS ubiquitination, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14days, 0.5μl/h) through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion increased baseline mean arterial pressure (126 ± 9* vs. 84 ± 4 mmHg) and renal sympathetic nerve activity (20.5 ± 2.3* vs. 6.4 ± 1.9 % of Max. Activity). Ang II infusion increased the expression of PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) with a concomitant 52% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, Ang II-mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX (protein synthesis inhibitor) 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. Substrate-dependent ligase 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). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO on pre-autonomic neurons in the PVN resulting in an increase in sympathetic outflow.

E3 Ubiquitin-Protein Ligase SMURF1 in the Nucleus Accumbens Mediates Cocaine Seeking

BackgroundSubstance use disorder is a neurobiological disease characterized by episodes of relapse despite periods of withdrawal. It is thought that neuroadaptations in discrete brain areas of the reward pathway, including the nucleus accumbens, underlie these aberrant behaviors. The ubiquitin–proteasome system degrades proteins and has been shown to be involved in cocaine-induced plasticity, but the role of E3 ubiquitin ligases, which conjugate ubiquitin to substrates, is unknown. Here, we examined E3 ubiquitin-protein ligase SMURF1 (SMURF1) in neuroadaptations and relapse behavior during withdrawal following cocaine self-administration. MethodsSMURF1 and downstream targets ras homolog gene family, member A (RhoA), SMAD1/5, and Runt-related transcript factor 2 were examined using Western blotting (n = 9–11/group), quantitative polymerase chain reaction (n = 6–9/group), co-immunoprecipitation (n = 9–11/group), tandem ubiquitin binding entities affinity purification (n = 5–6/group), and quantitative chromatin immunoprecipitation (n = 3–6/group) (2 rats/sample). Viral-mediated gene transfer (n = 7–12/group) and intra-accumbal microinjections (n = 9–10/group) were used to examine causal roles of SMURF1 and substrate RhoA, respectively, in cue-induced cocaine seeking. ResultsSMURF1 protein expression was decreased, while SMURF1 substrates RhoA and SMAD1/5 were increased, in the nucleus accumbens on withdrawal day 7, but not on withdrawal day 1, following cocaine self-administration. Viral-mediated gene transfer of Smurf1 or constitutive activation of RhoA attenuated cue-induced cocaine seeking, while catalytically inactive Smurf1 enhanced cocaine seeking. Furthermore, SMURF1-regulated, SMAD1/5-associated transcription factor Runt-related transcript factor 2 displayed increased binding at promoter regions of genes previously associated with cocaine-induced plasticity. ConclusionsSMURF1 is a key mediator of neuroadaptations in the nucleus accumbens following cocaine exposure and mediates cue-induced cocaine seeking during withdrawal.

Abstract 3539: Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity

Abstract Cells regulate their internal functions by the ubiquitylation of many key proteins, especially onco-proteins and tumor suppressors. As a result, ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anti-cancer targets. However, traditional methods for monitoring ubiquitylation patterns, including by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, are either insensitive, low throughput, or highly intensive. Here, we describe three Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiTest, HT-UbiTest, and UbiQuant S, which provide a platform to efficiently detect ubiquitylation of anti-cancer targets. UbiTest is an immunoblot-based assay, which can be used to easily identify ubiquitlyated proteins. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. HT-UbiTest extends this concept using a TUBE pulldown followed by two-antibody detection in a 96-well plate, and allows high-throughput absolute quantification of a target of interest. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in cells or tissue. Ubiquitylated proteins are identified by energy transfer between AlphaLISA bead-labeled TUBE and antibody against the endogenous protein of interest in a homogenous plate-based assay. Furthermore, we have extended these technologies to identify K63- and K48-linked poly-ubiquitylated proteins using poly-ubiquitin linkage-selective TUBEs. Several prominent therapeutic targets in oncology and immuno-oncology have been used to verify these assays, including Cbl-b and its substrates, p53, and USP7. The efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples using DUB and ubiquitin ligase inhibitors illustrate the power of this system. These novel assays are versatile tools for understanding the role of ubiquitylation in cancer treatment, and provide platforms to monitor disease biomarkers in response to drug action. Citation Format: Peter K. Foote, Xiaolong Lu, Rajesh Singh. Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3539.

CGMP Stimulates K48‐linked Poly‐ubiquitination of NKCC2 in Rats Thick Ascending Limb

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We showed that NKCC2 is constitutively ubiquitinated and degraded by the proteasome, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification in which the C‐terminus of the ubiquitin is attached to a substrate protein. Poly‐ubiquitination occurs when the C‐terminus of another ubiquitin, is then linked to the previously added ubiquitin, creating a chain. There are 7 lysine (k) in the ubiquitin molecule where poly‐ubiquitination can occur. K48‐linked poly‐ubiquitination on ubiquitin is associated with proteasomal degradation. Therefore, we hypothesized what cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2 in rats TAL. To test our hypothesis we immunoprecipitated K48‐linked poly‐ubiquitin using Tandem Ubiquitin Binding Entities (TUBEs) and western blot against NKCC2 in Sprague‐Dawley rat TAL suspensions. First, we performed a k48‐TUBEs concentration‐response curve against K48‐linked poly‐ubiquitination of NKCC2 to ensure linear relationship. TALs suspension were aliquoted in 4 samples and placed at 37°C for 30 minutes in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. TALs were lysed at 4°C and excess amount of protein lysate (150 μg) was incubated with increasing concentration of k48‐TUBEs. We obtained a linear increase in K48‐linked poly‐ubiquitination of NKCC2 with increasing concentration of k48‐TUBEs. Next, to study the effect of cGMP on k48‐ubiquitination of NKCC2. The conditions were set to fit within the linear range (80 μg of protein) to ensure complete immunoprecipitation of K48‐linked poly‐ubiquitination of NKCC2. We found that the membrane‐permeant cGMP analogue dibutiryl cGMP (db‐cGMP) stimulates K48‐ubiquitination on NKCC2 in a concentration‐dependent manner (baseline: 100.0%; db‐cGMP 100 μM: 115.3 ± 3.7%; db‐cGMP 250 μM: 127.8 ± 5.2%; db‐cGMP 500 μM: 148.3 ± 18.2% p<0.05). These data indicates that cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2, and suggest a triggering mechanism for NKCC2 degradation by the proteasome. We conclude that cGMP increases K48‐linked poly‐ubiquitination of NKCC2. To our knowledge, this is the first piece of evidence showing that cGMP may trigger a signal for NKCC2 degradation through stimulation of k48‐ubiquitination.Support or Funding InformationAHA Post‐Doctoral (14POST20110009)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.