E3 Ubiquitin Research Articles

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Abstract 2571: DBC1, a novel CBP-interacting protein, promotes p53 stability by regulating CBP-dependent p53 polyubiquitination

Abstract The acetyltransferase CBP, in conjunction with the E3 ubiquitin ligase enzyme, Mdm2, maintains physiologic levels of the tumor suppressor protein p53 in the absence of cellular stress, via a cytoplasmic, but not nuclear, p53-directed E4 polyubiquitin activity. CBP also possesses cytoplasmic, but not nuclear, E3 ubiquitin ligase, autoubiquitination activity. To understand the mechanism by which the ubiquitin ligase activities of CBP are compartmentalized in the cell, we have employed Multidimensional Protein Identification Technology (MudPIT) to identify cytoplasmic and nuclear CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP- interacting protein in both the nucleus and cytoplasm. The N-terminus of DBC1 bound to multiple CBP domains, including the putative E3/E4 domain of CBP located at the N-terminus. Interaction between DBC1 and CBP suppressed the in vitro E3 autoubiquitination activity of CBP. Functional studies demonstrated that DBC1 directly regulates the cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitin ligase activities. Knockdown of DBC1 in U2OS cells stimulated normally absent nuclear p53 polyubiquitination, and also caused a decrease in p53-dependent and p53-independent apoptosis, rescued by CBP/DBC1 double knockdown. Further, over-expression of DBC1 rescued the increase in nuclear p53 polyubiquitination and decrease in p53 half-life observed in DBC1 depleted cells. Together, these results identify DBC1 as a novel binding partner of CBP and a critical regulator of CBP ubiquitin ligase activities. Based on our data that DBC1 loss inactivates p53 function through destabilization, we queried the TCGA database for co-occurrence of DBC1 loss with maintenance of wild-type p53 status which showed that 33% of breast, 40% of lung, and 96% of prostate cancer cases with DBC1 alterations maintained wild-type p53 status. Therefore, by restoring DBC1 function, this work could lead to reactivation of p53 tumor suppressor activity in tumors with DBC1 alterations maintaining wild-type p53 status. Citation Format: Oluwatoyin E. Akande, Priyadarshan K. Damle, Nicholas E. Sherman, Steven R. Grossman. DBC1, a novel CBP-interacting protein, promotes p53 stability by regulating CBP-dependent p53 polyubiquitination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2571. doi:10.1158/1538-7445.AM2017-2571

Abstract 4913: Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation

Abstract Metastatic melanoma is highly aggressively and often therapy resistant. Signaling pathways which promote invasion of metastatic melanoma, also promote therapy resistance. Therapy resistant melanomas are characterized by high levels of the pro-invasive non-canonical Wnt molecule, Wnt5A. Previously, we described an adaptive stress response in highly invasive melanoma cells, which is characterized by a growth arrest and an increase in senescence markers, yet these cells retain the ability to invade and form colonies. These highly invasive cells do not undergo apoptosis following treatment with DNA damaging agents such as doxorubicin and are resistant to BRAFV600E targeted therapy. Recently, Lukin et al. have shown that p53 expression promotes survival of colorectal cancer cells via p21 expression and a reversible cell cycle arrest, allowing for repair of damaged DNA. Here, we show that highly invasive and therapy resistant melanoma cells express Wnt5A, p53 and p21, which regulate proliferation and slow cycling in invasive melanoma cells. The expression of p53 is promoted by Wnt5A, knock down of Wnt5A decreases p53 and p21 expression in these cells and decreases the number of cells arrested in G2/M following DNA damage. The cell cycle and apoptotic functions of p53 are highly regulated. iASPP, MDMX, and MDM2 have been shown to regulate the function of wild type p53 in melanoma cells. MDM2, an E4 ubiquitin ligase, regulates p53 by shuttling it out of the nucleus and targeting it for proteasomal degradation. We found that MDM2 expression increases in invasive melanoma cells following DNA damage, however, in these cells it is phosphorylated at serine 395. Phosphorylation of MDM2 at ser395 blocks its ability to export p53 from the nucleus, leading to increased p53 expression. Even with increased p53 expression, invasive melanoma cells undergo a cell cycle arrest following stress instead of apoptosis. The apoptotic function of p53 has been shown to be inhibited by nuclear iASPP, which is enriched in metastatic melanoma. We found that knocking down Wnt5A in invasive melanoma cells decreases the expression of iASPP. These data suggest that Wnt5A promotes MDM2 Ser395 phosphorylation and iASPP expression, blocking down regulation of p53 and its apoptotic function, while promoting cell cycle arrest and survival following stress. These data may reveal a mechanism by which highly invasive melanoma cells evade therapy to form therapy resistant sub-colonies at distant sites. Citation Format: Marie R. Webster, Amanpreet Kaur, Abibatou Ndoye, Curtis Kugel, Subhasree Basu, Alexander Valiga, Jessica Appleton, Ying-Jie Wang, Maureen Murphy, Ashani T. Weeraratna. Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2017-4913

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl small ubiquitin-like modifier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics.

XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination.

The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Inhibitors of ubiquitin E3 ligase as potential new antimalarial drug leads

BackgroundProtein ubiquitylation is an important post-translational regulation, which has been shown to be necessary for life cycle progression and survival of Plasmodium falciparum. Ubiquitin is a highly conserved 76 amino acid polypeptide, which attaches covalently to target proteins through combined action of three classes of enzymes namely, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). Ubiquitin E1 and E2 are highly conserved within eukaryotes. However, the P. falciparum E3 ligase is substantially variable and divergent compared to the homologs from other eukaryotes, which make the E3 ligase a parasite-specific target.MethodsA set of selected E3 ubiquitin ligase inhibitors was tested in vitro against a chloroquine-sensitive P. falciparum D6 strain (PfD6) and a chloroquine-resistant P. falciparum W2 strain (PfW2). The inhibitors were also tested against Vero and transformed THP1 cells for cytotoxicity. The lead antimalarial E3 ubiquitin ligase inhibitors were further evaluated for the stage-specific antimalarial action and effects on cellular development of P. falciparum in vitro. Statistics analysis was done by two-way ANOVA followed by Tukey and Sidak multiple comparison test using GraphPad Prism 6.ResultsE3 ligase inhibitors namely, JNJ 26854165, HLI 373 and Nutlin 3 showed prominent antimalarial activity against PfD6 and PfW2. These inhibitors were considerably less cytotoxic to mammalian Vero cells. JNJ 26854165, HLI 373 and Nutlin 3 blocked the development of P. falciparum parasite at the trophozoite and schizont stages, resulting in accumulation of distorted trophozoites and immature schizonts.ConclusionsInterruption of trophozoites and schizont maturation by the antimalarial E3 ligase inhibitors suggest the role of ubiquitin/proteasome functions in the intraerythrocytic development of malaria parasite. The ubiquitin/proteasome functions may be critical for schizont maturation. Further investigations on the lead E3 ligase inhibitors shall provide better understanding regarding the importance of E3 ligase functions in the malaria parasite as a potential new antimalarial drug target and a new class of antimalarial drug leads.

Dissecting the Specificity of Adenosyl Sulfamate Inhibitors Targeting the Ubiquitin-Activating Enzyme

Targeting the activating enzymes (E1) of ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) has emerged as a promising anti-cancer strategy, possibly overcoming the ineffectiveness of proteasome inhibitors against solid tumors. Here, we report crystal structures of the yeast ubiquitin E1 (Uba1) with three adenosyl sulfamate inhibitors exhibiting different E1 specificities, which are all covalently linked to ubiquitin. The structures illustrate how the chemically diverse inhibitors are accommodated within the adenylation active site. When compared with the previously reported structures of various E1 enzymes, our structures provide the basis of the preferences of these inhibitors for different Ub/Ubl-activating enzymes. Invitro inhibition assays and molecular dynamics simulations validated the specificities of the inhibitors as deduced from the structures. Taken together, the structures establish a framework for the development of additional compounds targeting E1 enzymes, which will display higher potency and selectivity.

Progress of nitrogen-phosphorus bond biosynthesis study of naturally occurring phosphoramidates

Naturally occurring phosphoramidates, which contain nitrogen-phosphorus (N−P) bonds, show high structure similarity with cytoplasmic carboxylates and phosphates. Phosphoramidates exhibit wide bioactivity spectrum attributed to the role as inhibitor of certain necessary enzymes in cell cycle. Three N−P bond biosynthetic pathways, which are respectively catalyzed by MccB, adenylyl sulphate: ammonia adenylyltransferase (APSAT) and Pyruvate phosphate dikinase (PPDK) homologues, are reviewed here. (1) MccB, a homologue of E1 ubiquitin ligase, catalyzed the phosphorylation of the asparagine residue of Microcin C7 precursor peptide. It consumed one molecule of ATP for the cyclization of the asparagine residue to give a succinimide. Then, the succinimidyl nitrogen atom attacked the Pα of a second ATP molecule to create an adenosine monophosphate- N -succinimide moiety. Agrocin 84, dinogunellin and phosmidosine shared identical AMP-NHR structural moiety with Microcin C7. Unfortunately, the biosynthetic gene clusters of dinogunellin and phosmidosine have not been identified yet. Agrocin 84 contains two N-P bonds, the first one linked the amino group of 2,3-dihydroxy-4-methyl-pentanamide with the phosphate group of 3′dAMP, and the second one bridged the amino group of the 3′dAMP purine ring with the phosphate group on the phosphorylated D-glucofuranosyloxy- phosphoryl. Analysis of Agrocin 84 biosynthetic gene cluster showed that agnA is homologous to mccB gene. Based on structural similarity and gene homology, it was proposed that the first N−P bond biosynthetic pathway of agrocin 84 is similar to that of Microcin C7. (2) APSAT catalyzed ammonolysis of adenosine-5′-phosphosulfate to give adenosine- 5′-phosphoramidate (AMP-NH2) and sulfate. AMP-NH2 was discovered from the extracts of Chlorella pyrenoidosa , Dictyostelium discoideum , Euglena gracilis var. bacillaris , spinach, barley and Escherichia coli . This APSAT protein was isolated from Chlorella pyrenoidosa and the biochemical characters were investigated. To eukaryotes, human Fhit (Fragile Histidine Triad) showed adenylyltransferase activity as well. It not only catalyzed the hydrolysis of diadenosine triphosphate (ApppA) to form AMP and ADP, but also catalyzed the ammonolysis of ApppA to form AMP-NH2 and ADP. In the hydrolysis of ApppA to AMP and ADP catalyzed by Fhit, first, ApppA and Mg2+ covalently bound to the 96th histidine residue of Fhit to form Fhit-AMP covalent intermediate and released ADP; then Fhit-AMP was further hydrolyzed to form AMP and Fhit. The Fhit-AMP could also be aminolyzed to give AMP-NH2. A possible mechanism for Fhit-catalysed ammonolysis involving nucleophilic attacks of NH3 to the covalent histidine-bound enzyme- nucleosidyl intermediate. (3) PPDK catalyzed the phosphorylation of pyruvate with an intermediate in which a phosphate group was loaded on the conserved histidine residue of PPDK. PPDK homologues PhtL and AgnD were proposed to catalyze the N-P bonds formation of Phaseolotoxin and Agrocin 84, respectively. The Walsh group of Harvard University have hypothesized that the Phaseolotoxin N-P bond was formed by nucleophilic attack of ornithine free amino to the phosphate group attached to the histidine residue of PhtL. Similarly, it can be assumed that the amino group of 3′dAMP purine ring attacking the phosphate group of AgnD1 (or AgnD2) histidine residue, makes the second N-P bond of Agrocin 84. This is a novel hypothesis of the biosynthesis of the N-P bonds in naturally occurring phosphoramidates, while the detailed enzymatic reactions remain to be studied further.

MicroRNA-1301 suppresses tumor cell migration and invasion by targeting the p53/UBE4B pathway in multiple human cancer cells

The p53 protein plays a critical role in preventing tumor development. Although numerous factors have been shown to directly or indirectly regulate p53, the mechanism of how microRNAs (miRNAs) modulate p53 remains unclear. Here, we identified miR-1301, a microRNA that regulates the activity and function of p53, by directly targeting the ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase. Notably, ectopic expression of miR-1301 inhibits dissemination and metastasis of tumor cells in a p53-dependent manner. Depletion of miR-1301 downregulates p53 function. Our results reveal that there is an inverse correlation between miR-1301 and UBE4B expression and p53 status in prostate cancer. Furthermore, low miR-1301 expression is often associated with incomplete methylation of its gene in human prostate tumors. Together, our results provide the first report indicating that miR-1301 functions as a tumor suppressor that inhibits tumor cell migration and invasion in multiple human cancer cells by regulating the UBE4B-p53 pathway.

Nicotine induces H9C2 cell apoptosis via Akt protein degradation.

Smoking is highly associated with cardiovascular diseases. However, the effect of nicotine, a key ingredient in smoking products, on cardiomyocyte apoptosis remains controversial. The present study aims to clarify the role of nicotine on cardiomyocyte cell apoptosis and to investigate the underlying mechanism. In the present study, H9C2 cells were exposed to nicotine at various concentrations (0, 10 and 100µM) for 48h. Cell Counting Kit‑8 and TUNEL assays were performed to assess cell viability and apoptosis, respectively, and reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to investigate the mRNA and protein expression. PYR‑41, a ubiquitin E1 inhibitor, was employed to investigate whether the ubiquitin‑proteasome system was involved in the downregulation of Akt. An Akt1 overexpression plasmid was used to demonstrate the role of Akt in H9C2 cells apoptosis. Tetratricopeptide repeat domain3 (TTC3) small interfering RNA (siRNA) was used to investigate the effect of TTC3 on Akt protein degradation. The results demonstrated that nicotine induced apoptosis in H9C2 cells compared with control cells (P<0.05). The protein level of Akt was downregulated by nicotine in a concentration‑dependent manner (P<0.05). PYR‑41 treatment restored the protein level of Akt. The cell viability was significantly improved by Akt overexpression when cells were exposed to nicotine at 10µM, compared with control cells. Nicotine also upregulated the level of TTC3 mRNA (P<0.05) and the protein level of Akt, and cell viability was recovered by TTC3 siRNA. In conclusion, the current study demonstrated that nicotine induced H9C2 cell apoptosis via Akt protein degradation, which may be mediated by TTC3.

Identifying Regulatory Posttranslational Modifications of PD-L1: A Focus on Monoubiquitinaton

A set of high-affinity, high-specificity posttranslational modification (PTM) enrichment tools was developed to generate an unbiased snapshot of four key PTM profiles (tyrosine phosphorylation, acetylation, ubiquitination, and SUMOylation 2/3) for the clinically important protein programmed cell death ligand 1 (PD-L1). The results showed that epidermal growth factor (EGF) treatment induced tyrosine phosphorylation, acetylation, and ubiquitination of PD-L1. Further characterization of EGF-induced PD-L1 ubiquitination revealed a significant increase in mono- and multiubiquitination of PD-L1 that occurred on glycosylated PD-L1. EGF induced mono- and multiubiquitination of PD-L1 preceded EGF-induced increases in PD-L1 protein levels. Chemical inhibitors of the EGFR pathway, gefitnib and SCH772984, suppressed PD-L1 mono- and multiubiquitination, and inhibition of the ubiquitin E1 activating enzyme, with the chemical inhibitor PYR41, was sufficient to block EGF-stimulated increases in PD-L1 protein levels. This study highlights the significance of identifying novel PTMs for PD-L1 and reveals potentially critical regulatory mechanisms that may be valuable therapeutic targets. In a broader context, this report validates an approach whereby one can gain insight into novel mechanisms of action by a simple and unbiased analysis of a PTM profile of potentially any endogenous protein of interest.

The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S.pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.

TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.

Mcl-1 is a Gate Keeper Regulating Cell Death in Cancer Cells

Selectively inducing cancer cells to death is the goal of cancer therapy. The discovery of B-cell lymphoma 2 (Bcl-2) family members regulating apoptotic cell death of cancer cells revealed new targets for cancer therapy. Bcl-2 family members can be classified into pro-apoptotic members and anti-apoptotic members among which myeloid cell leukemia 1 (Mcl-1) plays unique roles in regulating cell death and survival in cancer cells. Mcl-1 has a short half-life due to its degradation by multiple E3 ubiquitin-ligases. Under hypoxic conditions, Mcl-1 is up-regulated by activation of growth factor receptor, EGFR promoting cell survival whereas, prolonged/severe hypoxia leads to deactivation of EGFR and Mcl-1 degradation by E3 ubiquitin -ligase FBW7 contributing to cell death. Furthermore, cancer cells upregulated Mcl-1 contributing resistance to chemotherapeutic treatment such as by inhibitors of other antiapoptotic Bcl-2 members Bcl-2, Bcl-xL and Bcl-w. Therefore, Mcl- 1 might be the key player in pro-survival Bcl-2 family members in regulating cancer cell death. This encourages the on-going active development of Mcl-1 specific inhibitors for cancer treatment.

An E2-guided E3 Screen Identifies the RNF17-UBE2U Pair as Regulator of the Radiosensitivity, Immunodeficiency, Dysmorphic Features, and Learning Difficulties (RIDDLE) Syndrome Protein RNF168

Protein ubiquitination has emerged as a pivotal regulatory reaction that promotes cellular responses to DNA damage. With a goal to delineate the DNA damage signal transduction cascade, we systematically analyzed the human E2 ubiquitin- and ubiquitin-like-conjugating enzymes for their ability to mobilize the DNA damage marker 53BP1 onto ionizing radiation-induced DNA double strand breaks. An RNAi-based screen identified UBE2U as a candidate regulator of chromatin responses at double strand breaks. Further mining of the UBE2U interactome uncovered its cognate E3 RNF17 as a novel factor that, via the radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome protein RNF168, enforces DNA damage responses. Our screen allowed us to uncover new players in the mammalian DNA damage response and highlights the instrumental roles of ubiquitin machineries in promoting cell responses to genotoxic stress.

The Atypical Ubiquitin E2 Conjugase UBE2L3 Is an Indirect Caspase-1 Target and Controls IL-1β Secretion by Inflammasomes

SummaryCaspase-1 activation by inflammasome signaling scaffolds initiates inflammation and antimicrobial responses. Caspase-1 proteolytically converts newly induced pro-interleukin 1 beta (IL-1β) into its mature form and directs its secretion, triggering pyroptosis and release of non-substrate alarmins such as interleukin 1 alpha (IL-1α) and HMGB1. While some caspase-1 substrates involved in these events are known, the identities and roles of non-proteolytic targets remain unknown. Here, we use unbiased proteomics to show that the UBE2L3 ubiquitin conjugase is an indirect target of caspase-1. Caspase-1, but not caspase-4, controls pyroptosis- and ubiquitin-independent proteasomal degradation of UBE2L3 upon canonical and non-canonical inflammasome activation by sterile danger signals and bacterial infection. Mechanistically, UBE2L3 acts post-translationally to promote K48-ubiquitylation and turnover of pro-IL-1β and dampen mature-IL-1β production. UBE2L3 depletion increases pro-IL-1β levels and mature-IL-1β secretion by inflammasomes. These findings regarding UBE2L3 as a molecular rheostat have implications for IL-1-driven pathology in hereditary fever syndromes and in autoinflammatory conditions associated with UBE2L3 polymorphisms.

Characterization of Lineage Vs. Context-Dependent Essential Genes in Multiple Myeloma Using Crispr/Cas9 Genome Editing

Multiple Myeloma (MM) remains an incurable malignancy in part because of an incomplete understanding of which genes are critically responsible for MM cell survival and proliferation. To address this unmet need, and building on our recent functional genomics studies with the CRISPR/Cas9 gene editing platform (ASH 2015; Int. MM Workshop, Rome 2015), we reasoned that quantification of sgRNA depletion in the absence of any treatment could identify genes essential for the survival or proliferation of MM cells and better define their role as candidate therapeutic targets. To this end, we transduced Cas9-expressing RPMI-8226 and MM.1S cells with the lentiviral genome-scale GeCKO pooled library of sgRNAs. After culture of these cell lines for 2, 6, 8 or 12 weeks without any treatment, we identified, based on next generation sequencing for the sgRNA sequences, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR=0.05, based on MAGECK algorithm) in Cas9+ cells compared to their initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 50 cell lines from other hematologic malignancies (leukemia, lymphoma) and from 8 different types of solid tumors. We identified 3 broad categories of essential genes in MM cells: a) core essential genes, with sgRNA depletion across the majority of MM and non-MM lines of our study, representing cellular processes critical for practically all lineages (e.g. genes involved in regulation of basic transcription factor complexes, ribosomal function, proteasome, spliceosome, structural proteins for mitochondria and other key organelles, et.c.); b) genes selectively essential for MM cell lines, but not for the overwhelming majority of leukemia, lymphoma or solid tumor cell lines; c) genes with a role in small subset(s) of cell lines, across diseases, which harbor defined genetic features correlating with this dependency. We integrated our CRISPR/Cas9-based data on MM-selective essential genes with a reanalysis of the Achilles Heel shRNA screen in MM and non-MM cell lines (10 and 493, respectively) of the Cell Line Encyclopedia Program (CCLE) program. We applied a series of statistical tests (e.g. Wilcoxon rank test or marker selection feature of GENE-E algorithm with 1000 permutation tests) to identify genes with a significantly lower rank in sgRNA or shRNA depletion in MM vs. non-MM cell lines, across different specific thresholds for fold change and statistical significance. We identified more than 50 high-value candidate target genes with preferential essentiality in MM, compared to non-MM cell lines of diverse lineages. Prominent examples of such MM-selective, essential genes included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); otherNF-kB-related genes (e.g. IKBKB); PIM2 (but not PIM1 or PIM3 in this cell line panel); regulators of protein homeostasis, including diverse E2 and E3 ubiquitin ligases; and several other known or biologically-plausible dependencies which are under further evaluation. Many of these MM-selective dependencies exhibited significantly higher expression in MM, compared to non-MM cells, both in cell lines (based on the CCLE dataset) and patient-derived samples (comparison of Broad/MMRF vs. TCGA datasets, respectively). Notable observations of context-dependent essential genes include ARID1A in MM.1S cells (plausibly due to deficiency in its paralog ARID1B); and cases of both MM and non-MM cells with RAS mutations but lack of dependency on that gene. Targeting of lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our integrated genome-wide CRISPR/Cas9 and shRNA analyses in molecularly annotated panels of MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of UBE2N As a Therapeutic Approach in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Hematopoietic stem and progenitor cells (HSPC) from MDS and AML patients exhibit overexpression of TRAF6 and related innate immune pathway genes, suggesting a dependency of leukemic HSPC on activated innate immune signaling. Unfortunately, inhibiting TRAF6 directly has proven difficult, as few binding pockets on TRAF6 exist for small molecule targeting. UBE2N/Ubc13, a cofactor of TRAF6 and key enzyme in innate immune signaling, is an ubiquitin-conjugating E2 enzyme that catalyzes lysine 63 (K63)-linked ubiquitin chains on TRAF6 and its substrates. Importantly, a commercially available compound and our own chemical series of UBE2N inhibitors are available. In this study we evaluated the cellular and molecular effects of pharmacologic and genetic inhibition of UBE2N in MDS and AML cells.Pharmacologic inhibition of UBE2N with NSC697923 or genetic inhibition with shRNAs reduced the clonogenic capacity of MDSL/AML cell lines and primary cells while not significantly affecting normal HSPC. Treatment of MDS/AML cells with NSC697923 reduced the cellular metabolic activity, induced a G2/M cell cycle arrest, and increased cell death. Moreover, xenotransplantation of an MDS-derived patient cell line (MDSL) into immunodeficient mice (NSG-SGM3) showed a 50-70% reduced graft upon UBE2N knockdown relative to a non-silencing control. The cellular effects of UBE2N inhibition correspond with suppression of TRAF6-induced NF-kB activation of target genes. In addition, we found that NSC697923 treatment results in a dramatic loss of TRAF6 protein expression, which is rescued by inhibition of the proteasome. Intriguingly, our molecular analysis revealed that UBE2N inhibition shifts the stoichiometry of TRAF6 ubiquitin chains from K63-linked to K48-linked ubiquitin, resulting in proteasome-mediated degradation.To identify the molecular basis of UBE2N inhibition, we performed a global ubiquitin screen for changes in ubiquitinated substrates and gene expression profiling by RNA sequencing. For the ubiquitin screen, K63 ubiquitinated proteins were immunoprecipitated from MDSL cells upon pharmacologic inhibition of UBE2N, followed by mass spectrometry analysis. UBE2N inhibition significantly altered the ubiquitination of ~140 proteins involved in innate immune signaling, glycolysis, cell survival, RNA splicing, and DNA damage response. In parallel, RNA sequencing of MDSL cells treated with NSC697923 revealed expression changes in genes involved in mRNA processing, cell cycle and glycolysis. Several components of the E3 ligase anaphase-promoting complex APC/CDC20 were downregulated after UBE2N inhibition. As expected, increased expression of APC/CDC20 substrates (i.e., cyclin B1) were observed following treatment with NSC697923, suggesting that UBE2N inhibition in MDS/AML blocks degradation of APC/CDC20 targets and leads to mitotic alterations and apoptosis.One substrate identified in NSC697923-treated MDSL cells by the ubiquitin screen is DDB1, a component of the CUL4-CRBN E3 ligase complex targeted by Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and NSC697923 on MDS/AML cells. As compared to individual treatments, the combination of LEN and NSC697923 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and NSC697923 impaired MDS/AML cells that are refractory to treatment of LEN or NSC697923 alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS/AML.In summary, our data reveal a novel therapeutic target, an E2 ubiquitin conjugating enzyme (UBE2N), in MDS/AML. UBE2N inhibition suppresses the function and viability of MDS/AML cell lines and patient samples, due in part to degradation of TRAF6, suppressing innate immune signaling, and inducing mitotic alterations. Lastly, we show that inhibition of UBE2N alters ubiquitination of DDB1, a component of the CRBN complex, and cooperates with LEN to target MDS/AML cells. DisclosuresNo relevant conflicts of interest to declare.