Yi Sheng

EBNA1-mediated recruitment of a histone H2B deubiquitylating complex to the Epstein-Barr virus latent origin of DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.

A human ubiquitin conjugating enzyme (E2) - HECT E3 ligase structure-function screen

Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

CUL7 Is a Novel Antiapoptotic Oncogene

Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53. CUL7 binds directly to p53, and small interfering RNA-mediated knockdown of CUL7 results in the elevation of p53 protein levels. This antiapoptotic role of CUL7 enables this novel oncogene to cooperate with Myc to drive transformation. Deregulated ectopic expression of c-Myc and CUL7 promotes Rat1a cell growth in soft agar, and knockdown of CUL7 significantly blocks human neuroblastoma SHEP cell growth in an anchorage-independent manner. Furthermore, using public microarray data sets, we show that CUL7 mRNA is significantly overexpressed in non-small cell lung carcinoma and is associated with poor patient prognosis. We provide experimental evidence to show CUL7 is a new oncogene that cooperates with Myc in transformation by blocking Myc-induced apoptosis in a p53-dependent manner.

Structural basis of E2-25K/UBB+1 interaction leading to proteasome inhibition and neurotoxicity.

E2–25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB+1) and has been identified as a crucial factor regulating amyloid-β neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2–25K-UBB+1 interaction, we determined the three-dimensional structures of UBB+1, E2–25K and the E2–25K/ubiquitin, and E2–25K/UBB+1 complex. The structures revealed that ubiquitin or UBB+1 is bound to E2–25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2–25K mutants showed that disrupting UBB+1 binding markedly diminishes synthesis of neurotoxic UBB+1-anchored polyubiquitin. These results suggest that the interaction between E2–25K and UBB+1 is critical for the synthesis and accumulation of UBB+1-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.

The E3 ubiquitin ligase Mule acts through the ATM–p53 axis to maintain B lymphocyte homeostasis

Genetic manipulation reveals that Mule is vital for B cell development, proliferation, and homeostasis as a result of its ability to regulate p53 and ATM.

Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögrens syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

Anti-Ro52 autoantibodies from patients with Sjögren's syndrome inhibit the E3 ligase activity of Ro52 by blocking the E3:E2 interface

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögrens syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

Structural and Functional Comparison of the RING Domains of Two p53 E3 Ligases, Mdm2 and Pirh2

The tumor suppressor p53 maintains genome stability and prevents malignant transformation by promoting cell cycle arrest and apoptosis. Both Mdm2 and Pirh2 have been shown to ubiquitylate p53 through their RING domains, thereby targeting p53 for proteasomal degradation. Using structural and functional analyses, here we show that the Pirh2 RING domain differs from the Mdm2 RING domain in its oligomeric state, surface charge distribution, and zinc coordination scheme. Pirh2 also possesses weaker E3 ligase activity toward p53 and directs ubiquitin to different residues on p53. NMR and mutagenesis studies suggest that whereas Pirh2 and Mdm2 share a conserved E2 binding site, the seven C-terminal residues of the Mdm2 RING directly contribute to Mdm2 E3 ligase activity, a feature unique to Mdm2 and absent in the Pirh2 RING domain. This comprehensive analysis of the Pirh2 and Mdm2 RING domains provides structural and mechanistic insight into p53 regulation by its E3 ligases.

USP7/HAUSP promotes the sequence-specific DNA binding activity of p53.

The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity.