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.     

Mammalian HECT ubiquitin-protein ligases: Biological and pathophysiological aspects

Members of the HECT family of E3 ubiquitin-protein ligases are characterized by a C-terminal HECT domain that catalyzes the covalent attachment of ubiquitin to substrate proteins and by N-terminal extensions of variable length and domain architecture that determine the substrate spectrum of a respective HECT E3. Since their discovery in 1995, it has become clear that deregulation of distinct HECT E3s plays an eminent role in human disease or disease-related processes including cancer, cardiovascular and neurological disorders, viral infections, and immune response. Thus, a detailed understanding of the structure–function aspects of HECT E3s as well as the identification and characterization of the substrates and regulators of HECT E3s is critical in developing new approaches in the treatment of respective diseases. In this review, we summarize what is currently known about mammalian HECT E3s, with a focus on their biological functions and roles in pathophysiology.This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

The Active Form of E6-associated protein (E6AP)/UBE3A Ubiquitin Ligase Is an Oligomer

Employing ¹²⁵I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508 Ų and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe⁷²⁷ and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases k_cat 62-fold but fails to affect E2∼ubiquitin binding or subsequent formation of the Hect domain Cys⁸²⁰∼ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe⁷²⁷-dependent trimer formation and catalytic activity (K_i = 12 mm), as does a conserved α-helical peptide corresponding to residues 474–490 of E6AP isoform 1 (K_i = 22 μm) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe⁷²⁷ intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (K_activation = 1.5 nm) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the k_cat defect of the Phe⁷²⁷ mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.     

Impaired Phosphorylation and Ubiquitination by p70 S6 Kinase (p70S6K) and Smad Ubiquitination Regulatory Factor 1 (Smurf1) Promote Tribbles Homolog 2 (TRIB2) Stability and Carcinogenic Property in Liver Cancer

Tribbles homolog 2 (TRIB2) is critical for both solid and non-solid malignancies. Recently, TRIB2 was identified as a liver cancer-specific Wnt/β-catenin signaling downstream target and is functionally important for liver cancer cell survival and transformation. TRIB2 functions as a protein that interacts with E3 ubiquitin ligases and thereby modulates protein stability of downstream effectors. However, the regulation underlying TRIB2 protein stability per se has not yet been reported. In this study, we found that TRIB2 was up-regulated and exhibited high stability in liver cancer cells compared with other cells. We performed a structure-function analysis of TRIB2 and identified a domain (amino acids 1–5) at the N terminus that interacted with the E3 ubiquitin ligase Smurf1 and was critical for protein stability. Deletion of this domain extended TRIB2 half-life time accompanied with a more significant malignant property compared with wild type TRIB2. Furthermore, Smurf1-mediated ubiquitination required phosphorylation of TRIB2 by p70 S6 kinase (p70S6K) via another domain (amino acids 69–85) that is also essential for correct TRIB2 subcellular localization. Mutation of Ser-83 diminished p70S6K-induced phosphorylation of TRIB2. Moreover, the high stability of TRIB2 may be due to the fact that both p70S6K and Smurf1 were down-regulated and negatively correlated with TRIB2 expression in both liver cancer tissues and established liver cancer cell lines. Taken together, impaired phosphorylation and ubiquitination by p70S6K and Smurf1 increase the protein stability of TRIB2 in liver cancer and thus may be helpful in the development of diagnosis and treatment strategies against this malignant disease.     

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

A Structural Element within the HUWE1 HECT Domain Modulates Self-ubiquitination and Substrate Ubiquitination Activities

E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases.     

Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold

SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.     

Flexible cullins in cullin-RING E3 ligases allosterically regulate ubiquitination

How do the cullins, with conserved structures, accommodate substrate-binding proteins with distinct shapes and sizes? Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from E2 to the substrate, tagging the substrate for degradation. They contain substrate-binding, adaptor, cullin, and Rbx proteins. Previously, we showed that substrate-binding and Rbx proteins are flexible. This allows shortening of the E2-substrate distance for initiation of ubiquitination or increasing the distance to accommodate the polyubiquitin chain. However, the role of the cullin remained unclear. Is cullin a rigid scaffold, or is it flexible and actively assists in the ubiquitin transfer reaction? Why are there different cullins, and how do these cullins specifically facilitate ubiquitination for different substrates? To answer these questions, we performed structural analysis and molecular dynamics simulations based on Cul1, Cul4A, and Cul5 crystal structures. Our results show that these three cullins are not rigid scaffolds but are flexible with conserved hinges in the N-terminal domain. However, the degrees of flexibilities are distinct among the different cullins. Of interest, Cul1 flexibility can also be changed by deletion of the long loop (which is absent in Cul4A) in the N-terminal domain, suggesting that the loop may have an allosteric functional role. In all three cases, these conformational changes increase the E2-substrate distance to a specific range to facilitate polyubiquitination, suggesting that rather than being inert scaffold proteins, cullins allosterically regulate ubiquitination.     

Mono-ubiquitination drives nuclear export of the human DCN1-like protein hDCNL1

Conjugation of Nedd8 to a cullin protein, termed neddylation, is an evolutionarily conserved process that functions to activate the cullin-RING family E3 ubiquitin ligases, leading to increased proteasomal degradation of a wide range of substrate proteins. Recent emerging evidence demonstrates that cellular neddylation requires the action of Dcn1, which, in humans, consists of five homologues designated as hDCNL1-5. Here we revealed a previously unknown mechanism that regulates hDCNL1. In cultured mammalian cells ectopically expressed hDCNL1 was mono-ubiquitinated predominantly at K143, K149, and K171. Using a classical chromatographic purification strategy, we identified Nedd4-1 as an E3 ligase that can catalyze mono-ubiquitination of hDCNL1 in a reconstituted ubiquitination system. In addition, the hDCNL1 N-terminal ubiquitin-binding domain is necessary and sufficient to mediate mono-ubiquitination. Finally, fluorescence microscopic and subcellular fractionation analyses revealed a role for mono-ubiquitination in driving nuclear export of hDCNL1. Taken together, these results suggest a mono-ubiquitination-mediated mechanism that governs nuclear-cytoplasmic trafficking of hDCNL1, thereby regulating hDCNL1-dependent activation of the cullin-RING E3 ubiquitin ligases in selected cellular compartments.     

The Ubiquitin-conjugating Enzyme (E2) Ube2w Ubiquitinates the N Terminus of Substrates

Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification, we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH₂ group as the site of conjugation. To confirm N-terminal ubiquitination, we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked, ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein Tau. Finally, we directly sequenced the N terminus of unmodified and ubiquitinated ataxin-3, demonstrating that Ube2w attaches ubiquitin to the N terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N terminus of substrates.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation

Mdm2 and Mdmx are oncoproteins that have essential yet nonredundant roles in development and function as part of a multicomponent ubiquitinating complex that targets p53 for proteasomal degradation. However, in response to DNA damage, Mdm2 and Mdmx are phosphorylated and protect p53 through various mechanisms. It has been predicted that Mdm2-Mdmx complex formation modulates Mdm2 ligase activity, yet the mechanism that promotes formation of Mdm2-Mdmx complexes is unknown. Here, we show that optimal Mdm2-Mdmx complex formation requires c-Abl phosphorylation of Mdm2 both in vitro and in vivo. In addition, Abl phosphorylation of Mdm2 is required for efficient ubiquitination of Mdmx in vitro, and eliminating c-Abl signaling, using c-Abl(-/-) knock-out murine embryonic fibroblasts, led to a decrease in Mdmx ubiquitination. Further, p53 levels are not induced as efficiently in c-Abl(-/-) murine embryonic fibroblasts following DNA damage. Overall, these results define a direct link between genotoxic stress-activated c-Abl kinase signaling and Mdm2-Mdmx complex formation. Our results add an important regulatory mechanism for the activation of p53 in response to DNA damage.     

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.     

Turning the RING Domain Protein MdmX into an Active Ubiquitin-Protein Ligase

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.     

Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders

The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross-functionality in additional neurodegenerative diseases, for instance, to eliminate polyglutamine-expanded proteins, reducing their aggregation and toxicity and reinstate proteasome function. The dual actions of CHIP (molecular co-chaperone and E3 ligase) and Parkin (as E3-ubiquitin ligase and anti-oxidant) may also play a role in suppressing inflammatory reactions in animal models of neurodegeneration. In this review, we focus on the significance of CHIP and Parkin as inducers of amyloid clearance, as cytoprotectants and in the suppression of reactive inflammation. A case is made for more effort to explore whether neurodegeneration associated with proteinopathies can be arrested at early stages by promoting their mutual action.