Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125

Retinoic acid-inducible gene I (RIG-I) plays a pivotal role in the regulation of cytokine production induced by pathogens. The RIG-I also augments the production of IFN and other cytokines via an amplification circuit. Because the production of cytokines is closely controlled, up- and down-regulation of RIG-I signaling also needs strict regulation. The mechanism of this regulation, however, remains elusive. Here, we found that RIG-I undergoes proteasomal degradation after conjugation to ubiquitin by RNF125. Further, RNF125 conjugates ubiquitin to MDA5, a family protein of RIG-I as well as IPS-1, which is also a downstream protein of RIG-I signaling that results in suppressing the functions of these proteins. Because RNF125 is enhanced by IFN, these functions constitute a negative regulatory loop circuit for IFN production.     

FRS2 alpha attenuates FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl

Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Here, we demonstrate that Grb2 bound to tyrosine-phosphorylated FRS2 alpha forms a ternary complex with Cbl by means of its Src homology 3 domains resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 alpha in response to FGF stimulation. These observations highlight the importance of FRS2 alpha in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2 alpha-/- cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms.     

Pruning and loss of excitatory synapses by the parkin ubiquitin ligase

Mutations in the PARK2 gene cause hereditary Parkinson disease (PD). The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD.     

A binding motif for Siah ubiquitin ligase

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.     

The ubiquitin ligase Nedd4-1 is dispensable for the regulation of PTEN stability and localization

PTEN is a tumor suppressor frequently mutated in cancer. Recent reports implicated Nedd4-1 as the E3 ubiquitin ligase for PTEN that regulates its stability and nuclear localization. We tested the physiological role of Nedd4-1 as a PTEN regulator by using cells and tissues derived from two independently generated strains of mice with their Nedd4-1 gene disrupted. PTEN stability and ubiquitination were indistinguishable between the wild-type and Nedd4-1-deficient cells, and an interaction between the two proteins could not be detected. Moreover, PTEN subcellular distribution, showing prominent cytoplasmic and nuclear staining, was independent of Nedd4-1 presence. Finally, activation of PKB/Akt, a major downstream target of cytoplasmic PTEN activity, and the ability of PTEN to transactivate the Rad51 promoter, a measure of its nuclear function, were unaffected by the loss of Nedd4-1. Taken together, our results fail to support a role for Nedd4-1 as the E3 ligase regulating PTEN stability and subcellular localization.     

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.Covalent modification of proteins by ubiquitin (Ub) has an impact on nearly all eukaryotic cell biology. The attachment of Ub is accomplished by three enzymes: an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase (1). In the final stage of the pathway, the E3 ligase facilitates transfer from a Ub-loaded E2 (termed an E2∼Ub conjugate) onto a substrate lysine. Minimally, functional E3s contain an E2-binding domain and a substrate-recognition domain, enabling them to bind an E2∼Ub and a substrate simultaneously. The majority of E3s harbor either a RING (really interesting new gene) domain or a related U-box domain to bind cognate E2s. RING-type E3s enhance transfer of Ub directly from an E2’s active site to a substrate lysine without an intermediate transfer step of Ub to the E3 itself, as occurs with homology to E6AP carboxyl-terminus (HECT)-type ligases. In addition to providing proximity between the Ub attached to the active site of an E2 and a substrate amino group, RING-type E3s activate E2∼Ub conjugates allosterically (2–11). Allosteric activation relies on promotion of catalytically active “closed” conformations of an E2∼Ub conjugate that presumably arrange the E2 active site thioester for access and attack by an incoming nucleophile. Thus, two processes contribute to the rate enhancement of Ub transfer by RING-type E3s: (i) proximity (and, in some cases, orientational) effects and (ii) promotion of reactive states of the E2∼Ub. Recent studies (8–10) have shown that minimal RING-type domains that lack a substrate-binding activity (and therefore cannot provide a proximity enhancement) are able to enhance the intrinsic reactivity of E2∼Ub species, demonstrating that the two sources of rate enhancement are separable and independent. Although substrate proximity effects are likely to be idiosyncratic for each E3/substrate pair, the mechanisms that underlie allosteric activation appear to be shared among E3s. Furthermore, allosteric activation of an E2∼Ub is necessary and sufficient for RING-type E3 ligase activity. Therefore, a more thorough understanding of the allosteric mechanism and its structural bases should prove insightful for a majority of Ub E3 ligases.Recent structural studies of RING and U-box domains with E2∼Ub moieties reveal common features: (i) a closed E2∼Ub conformation and (ii) an intermolecular hydrogen bond between a conserved E3 side chain and an E2 backbone carbonyl (2, 3, 11). Mutational analyses demonstrate that both are critical for E3 allosteric enhancement of Ub transfer (3). Despite these conserved features, E3 ligases display large differences in Ub transfer activity even when working with the same E2, suggesting that other, possibly more subtle, effects are in play. Traditional approaches for dissecting enzymatic processes involve detailed structural studies and targeted mutational analyses, but these approaches are limited by the number of mutants that can be analyzed. Typically, the mutations generated disrupt activity and are often restricted to protein/protein interfaces, thereby ignoring much of the possible sequence space. Such limitations prompted us to use a high-throughput method, known as “deep mutational scanning” (12, 13), that can assess the effects of over 10⁵ sequence variants of a single protein simultaneously. We adapted this method to generate a sequence–function map of the U-box of the murine E3 ligase ubiquitination factor E4B (Ube4b). The U-box domain of Ube4b is an ideal candidate for this approach because it functions as a monomer; its structure has been solved both in isolation and in complex with the E2 UbcH5c; and with an extended N-terminal region, it exhibits auto-ubiquitination activity in vitro (14, 15). In vivo, the human homolog UBE4B polyubiquitinates the tumor suppressor p53 to target it for degradation by the proteasome. Increased expression of UBE4B and amplification of the genomic locus of UBE4B have been found in patient-derived medulloblastoma tumors that also have lower levels of p53 (16), suggesting that increased UBE4B activity might be oncogenic by reducing p53 abundance.We used the E2 UbcH5c in reactions to assess the Ub ligase activity of nearly 100,000 unique protein variants of the Ube4b U-box domain, over 900 of which contained single amino acid substitutions. Although most single mutations decreased enzymatic activity, a few mutations greatly increased activity both in in vitro reactions and in promoting the degradation of p53 in a human cell line. We used NMR to characterize four activity-enhancing mutations and found two classes: One class enhances E2∼Ub catalysis by increasing E3:E2-binding affinity, and the other augments the allosteric capacity of the U-box to promote catalytically active E2∼Ub conformational states. These two classes of mutations are distinct, and mutations from both classes can be combined to enhance activity even further. Selecting for functional U-box variants in the presence of a different E2, Ube2w, resulted in a similar set of Ube4b-activating mutations, consistent with the hypothesis that mechanisms by which E3s allosterically activate intrinsic E2∼Ub reactivity are shared and that enhancing mutations will be generalizable. Additionally, we identified a Ube4b variant that has reduced activity with UbcH5c but retains activity with Ube2w, suggesting that it may be possible to use deep mutational strategies to identify and manipulate sources of E3:E2 specificity. Our deep mutational scans and structural analyses further define the molecular basis for E3-induced E2∼Ub allosteric activation and provide tools for future structural and functional studies of E3 ligases.     

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

The high affinity receptor for IgE, FcepsilonRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcepsilonRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcepsilonRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcepsilonRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcepsilonRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcepsilonRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcepsilonRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.     

A role for ubiquitin ligase recruitment in retrovirus release

Retroviral Gag polyproteins have specific regions, commonly referred to as late assembly (L) domains, which are required for the efficient separation of assembled virions from the host cell. The L domain of HIV-1 is in the C-terminal p6(gag) domain and contains an essential P(T/S)AP core motif that is widely conserved among lentiviruses. In contrast, the L domains of oncoretroviruses such as Rous sarcoma virus (RSV) have a more N-terminal location and a PPxY core motif. In the present study, we used chimeric Gag constructs to probe for L domain activity, and observed that the unrelated L domains of RSV and HIV-1 both induced the appearance of Gag-ubiquitin conjugates in virus-like particles (VLP). Furthermore, a single-amino acid substitution that abolished the activity of the RSV L domain in VLP release also abrogated its ability to induce Gag ubiquitination. Particularly robust Gag ubiquitination and enhancement of VLP release were observed in the presence of the candidate L domain of Ebola virus, which contains overlapping P(T/S)AP and PPxY motifs. The release defect of a minimal Gag construct could also be corrected through the attachment of a peptide that serves as a physiological docking site for the ubiquitin ligase Nedd4. Furthermore, VLP formation by a full-length Gag polyprotein was sensitive to lactacystin, which depletes the levels of free ubiquitin through inhibition of the proteasome. Our findings suggest that the engagement of the ubiquitin conjugation machinery by L domains plays a crucial role in the release of a diverse group of enveloped viruses.     

Association of NEDD4L ubiquitin ligase with essential hypertension

NEDD4L is a ubiquitin ligase that controls cell surface expression of kidney epithelial Na+ channels by ubiquitin-mediated endocytosis and lysosome targeting. Thus, it is a significant determinant of Na+ reabsorption in the distal nephron. The NEDD4L gene is located on human chromosome 18q21 within several blood pressure quantitative trait loci, including those for familial orthostatic hypotension, essential hypertension, pulse pressure, and systolic blood pressure response to postural challenge. Because of the importance of NEDD4L to Na+ balance, many of these studies have proposed that mutations in NEDD4L may be responsible for these blood pressure phenotypes. To test this hypothesis, we fine-mapped the NEDD4L region in 2 families with orthostatic hypotension, which we previously reported to be linked to human chromosome 18q21 but failed to implicate NEDD4L in these families. We also typed multiple NEDD4L single-nucleotide polymorphisms (SNPs) in a collection of US whites, Greek whites, and African-Americans individuals with essential hypertension. A significant association between several SNPs and hypertension was observed in all 3 populations. One of the SNPs associated in African Americans is known to result in premature truncation of the NEDD4L protein. Thus, genetic variation in NEDD4L may play a role in the development or progression of some forms of abnormal blood pressure.     

The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways

The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, our data establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms.     

Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase

We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the folding of its client proteins into their functional conformation. Many Hsp90 clients have been reported to be aberrantly expressed in a number of cancers. We demonstrate Cul5 interaction with members of the Hsp90 chaperone complex as well as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the site of ErbB2 at the plasma membrane and subsequent induction of polyubiquitination and proteasomal degradation. We also demonstrate Cul5 involvement in regulation of another Hsp90 client, Hif-1α. We observed Cul5 degradation of ErbB2 to occur independently of ElonginB-ElonginC function. The involvement of Cul5 in Hsp90 client regulation has implications in the effectiveness of Hsp90 targeted chemotherapy, which is currently undergoing clinical trials. The link between Cul5 and Hsp90 client regulation may represent an avenue for cancer drug development.     

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT–CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT^Δ69–83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.Studies with simian virus 40 (SV40), a member of the Polyomaviridae family of tumor viruses, have led to fundamental insights into molecular processes of cell transformation and oncogenesis (1, 2). SV40 encodes the large tumor antigen (LT) with the potential to transform cells in culture and induce tumors in rodents. The tumorigenic features of SV40 have been attributed to binding and deactivation of key tumor suppressor proteins of the host cell including p53 and members of the retinoblastoma (pRB) family (1–3). In addition, SV40 LT was shown to be physically associated with Cullin 7 (CUL7; also named p185 or p193) (4, 5) as well as insulin receptor substrate 1 (IRS1) (6). It has been proposed that the association of SV40 LT with either CUL7 or IRS1 is critical to SV40 oncogenic transformation (7–9). However, the functional effect of LT interaction with CUL7/IRS1 and their pathophysiological interrelation remains mostly unknown.CUL7 is a scaffold protein responsible for assembling the multisubunit Cullin-RING E3 ubiquitin ligase 7 (CRL7) that consists of the RING-finger protein ROC1 and the Skp1-Fbw8 substrate-targeting subunit (10, 11). Genetic studies documented a pivotal growth-regulatory role of CRL7. Both cul7 (12) and fbw8 (13) null mice exhibit intrauterine growth retardation. In addition, CUL7 germ-line mutations were linked to 3-M syndrome, a hereditary disorder characterized by pre- and postnatal growth retardation in humans (14, 15), as well as Yakut dwarfism syndrome (16). DeCaprio and colleagues mapped the CUL7 interaction domain on SV40 LT to residues 69–83 and demonstrated that the CUL7 binding-deficient deletion mutant (LT^Δ69–83) lost its transformation potential despite maintaining its ability to bind and inactivate p53 and pRB members (8, 9). This suggested that CUL7 may act as a tumor suppressor and that constraining growth-inhibitory functions of CRL7 may be critical to SV40 transformation.We previously identified IRS1, a component of the insulin and insulin-like growth factor 1 (IGF1) signaling pathway, as a proteolytic target of CRL7 (17). Binding of insulin or IGF1 to its receptor induces tyrosine phosphorylation of IRS1 and subsequent activation of phosphatidylinositol-3-kinase (PI3K)/AKT and Erk mitogen-activated pathway kinase (MAPK) pathways (18). It was shown that CRL7-induced degradation of IRS1 is part of a negative feedback loop via mechanistic target of rapamycin complex 1 (mTORC1) to restrain IRS1 downstream signaling (17, 19). A more recent study suggested an mTORC2-dependent feedback inhibition of IRS1 by direct phosphorylation of Fbw8, resulting in enhanced stability of this F-box protein that promotes IRS1 degradation (20). Collectively, these studies have implicated roles for CRL7 in regulating both mTORC1 and mTORC2 signaling. Based on the above observations, we investigated whether SV40 LT impacts on CRL7 feedback regulation of IRS1 signaling in addition to its effects on p53 and pRB members.     

Inhibitors of cullin-RING E3 ubiquitin ligase 4 with antitumor potential

Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4’s core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4’s substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds’ cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4–expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

Cullin-RING (really intersting new gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest RING-type E3 family, consisting of ∼300 members, ∼50% of the E3s identified in humans (1, 2). CRLs target many critical regulators of cell division and signaling. Canonical CRLs are modular complexes, in which a cullin (CUL) subunit’s N-terminal domain assembles interchangeably with CUL-specific substrate receptors capable of binding a substrate. On the other hand, a CUL’s C-terminal domain (CTD) binds a RING finger protein, ROC1/RBX1 for CUL1 to 4 or ROC2 for CUL5, to form a core ligase complex. CRLs’ core ligase collaborates with E2 Ub-conjugating enzymes for transferring Ub(s) to the bound substrate or a Ub moiety on a growing Ub chain.A selective small-molecule modulator of CRLs’ function allows us to address mechanistic and phenotypic questions about its targets in biochemical, cell-based, and animal studies. To date, there is only one Food and Drug Administration (FDA)-approved E3 drug class that targets the substrate receptor cereblon (thalidomide/lenalidomide) (3). Current drug/probe discovery efforts against the Ub-proteasome system depend heavily on traditional methods that exploit the ability of small-molecule agents to disable an enzyme’s catalytic pocket. However, RING-type E3s are atypical enzymes and contribute to ubiquitination by mediating protein–protein interactions with substrate, E2, and Ub (1). High-resolution structural studies have shown that interactions involving E3’s RING domain, E2, and Ub are characterized by large, relatively flat interfaces (4). Such perceived “undruggable” features impose a significant barrier to structure-based ligand search using either virtual or fragment-based physical screening.To date, there are no reported small-molecule lead compounds targeting the catalytic activity of any CRL. To address this need, we have recently created a novel high-throughput screen (HTS) platform using the fluorescence (Förster) resonance energy transfer (FRET) K48 di-Ub assay (5). In this system, a FRET signal is generated as a result of covalent conjugation of two Ub molecules carrying a pair of matching fluorophores in a reaction that requires E1, E2 Cdc34, and an E3 CRL1 subcomplex (ROC1–CUL1 CTD). Fully functional Ub variants were created to allow only one nucleophilic attack that produces a single Ub–Ub isopeptide bond, thereby eliminating the complexity associated with polyubiquitin chain assembly to ensure a high degree of reproducibility for effective HTS. Each fluorophore is placed to either donor or receptor Ub at a specific site in a manner that satisfies optimal energy transfer. A pilot HTS identified a small-molecule compound, suramin (an antitrypanosomal drug), that can inhibit E3 CRL1 activity by disrupting its ability to recruit E2 Cdc34 (5). These observations have provided proof-of-principle evidence that an E2–E3 interface can be perturbed through small-molecule modulators. The current study describes a large-scale HTS and extensive follow-up hit-to-lead studies, which identified a class of small-molecule inhibitors against E3 CRLs.     

E6AP ubiquitin ligase regulates PML-induced senescence in Myc-driven lymphomagenesis

Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for the E3 ubiquitin ligase E6AP in the regulation of promyelocytic leukemia protein (PML) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP-PML axis in B-cell lymphoma development. A partial loss of E6AP attenuated Myc-induced B-cell lymphomagenesis. This tumor suppressive action was achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP expressed elevated levels of PML and PML-nuclear bodies with a concomitant increase in markers of cellular senescence, including p21, H3K9me3, and p16. Consistently, PML deficiency accelerated the rate of Myc-induced B-cell lymphomagenesis. Importantly, E6AP expression was elevated in ～ 60% of human Burkitt lymphomas, and down-regulation of E6AP in B-lymphoma cells restored PML expression with a concurrent induction of cellular senescence in these cells. Our findings demonstrate that E6AP-mediated down-regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML observed in non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma.