The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer.

The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.

Sumoylation regulates protein dynamics during meiotic chromosome segregation in C. elegans oocytes.

Oocyte meiotic spindles in most species lack centrosomes and the mechanisms that underlie faithful chromosome segregation in acentrosomal meiotic spindles are not well understood. In C. elegans oocytes, spindle microtubules exert a poleward force on chromosomes that is dependent on the microtubule-stabilising protein CLS-2, the orthologue of the mammalian CLASP proteins. The checkpoint kinase BUB-1 and CLS-2 localise in the central spindle and display a dynamic localisation pattern throughout anaphase, but the signals regulating their anaphase-specific localisation remains unknown. We have shown previously that SUMO regulates BUB-1 localisation during metaphase I. Here, we found that SUMO modification of BUB-1 is regulated by the SUMO E3 ligase GEI-17 and the SUMO protease ULP-1. SUMO and GEI-17 are required for BUB-1 localisation between segregating chromosomes during early anaphase I. We also show that CLS-2 is subject to SUMO-mediated regulation; CLS-2 precociously localises in the midbivalent when either SUMO or GEI-17 are depleted. Overall, we provide evidence for a novel, SUMO-mediated control of protein dynamics during early anaphase I in oocytes.

A SUMO-Dependent Protein Network Regulates Chromosome Congression during Oocyte Meiosis.

During Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate in vivo. In vitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.

Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage.

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways.

Proteome-wide identification of SUMO modification sites by mass spectrometry.

The protein called 'small ubiquitin-like modifier' (SUMO) is post-translationally linked to target proteins at the ɛ-amino group of lysine residues. This 'SUMOylation' alters the behavior of the target protein, a change that is utilized to regulate diverse cellular processes. Understanding the target-specific consequences of SUMO modification requires knowledge of the location of conjugation sites, and we have developed a straightforward protocol for the proteome-wide identification of SUMO modification sites using mass spectrometry (MS). The approach described herein requires the expression of a mutant form of SUMO, in which the residue preceding the C-terminal Gly-Gly (diGly) is replaced with a lysine (SUMO(KGG)). Digestion of SUMO(KGG) protein conjugates with endoproteinase Lys-C yields a diGly motif attached to target lysines. Peptides containing this adduct are enriched using a diGly-Lys (K-ɛ-GG)-specific antibody and identified by MS. This diGly signature is characteristic of SUMO(KGG) conjugation alone, as no other ubiquitin-like protein (Ubl) yields this adduct upon Lys-C digestion. We have demonstrated the utility of the approach in SUMOylation studies, but, in principle, it may be adapted for the site-specific identification of proteins modified by any Ubl. Starting from cell lysis, this protocol can be completed in ∼5 d.

Structural basis for the RING-catalyzed synthesis of K63-linked ubiquitin chains.

RING E3 ligase-catalyzed formation of K63-linked ubiquitin chains by the Ube2V2-Ubc13 E2 complex is required in many important biological processes. Here we report the structure of the RING-domain dimer of rat RNF4 in complex with a human Ubc13∼Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with K63 in a position favorable for attack on the linkage between Ubc13 and the donor (second) ubiquitin held in the active 'folded back' conformation by the RING domain of RNF4. We verified the interfaces identified in the structure by in vitro ubiquitination assays of site-directed mutants. To our knowledge, this represents the first view of synthesis of K63-linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase-mediated catalysis.

Ubiquitin C-terminal hydrolases cleave isopeptide- and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers.

Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on Ub homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preference for N-terminal deubiquitylating activity as they are capable of cleaving N-terminal Ub from SUMO2 and Ube2W, while displaying no activity against any of the eight Ub linkage types. Surprisingly, this ability to cleave Ub from SUMO2 was 100 times more efficient for UCH-L3 when we deleted the unstructured N-terminus of SUMO2, demonstrating that UCH enzymes can cleave Ub from structured proteins. However, UCH-L3 could also cleave chemically synthesized isopeptide-linked Ub from lysine 11 (K11) of SUMO2 with similar efficiency, demonstrating that UCH DUB activity is not limited to peptide-linked Ub. These findings advance our understanding of the specificity of the UCH family of DUBs, which are strongly implicated in cancer and neurodegeneration but whose substrate preference has remained unclear. In addition, our findings suggest that the reversal of Ube2W-mediated N-terminal ubiquitylation may be one physiological role of UCH DUBs in vivo.

Proteome-wide identification of SUMO2 modification sites.

Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.

BC-box protein domain-related mechanism for VHL protein degradation.

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

Ube2W conjugates ubiquitin to α-amino groups of protein N-termini.

The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13-UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.

Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis.

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.

Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples.

Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LC-MS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.

Mechanism of ubiquitylation by dimeric RING ligase RNF4.

Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation, we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model, the 'Ile44 hydrophobic patch' of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING, and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.

Functional interactions between ubiquitin E2 enzymes and TRIM proteins.

The TRIM (tripartite motif) family of proteins is characterized by the presence of the tripartite motif module, composed of a RING domain, one or two B-box domains and a coiled-coil region. TRIM proteins are involved in many cellular processes and represent the largest subfamily of RING-containing putative ubiquitin E3 ligases. Whereas their role as E3 ubiquitin ligases has been presumed, and in several cases established, little is known about their specific interactions with the ubiquitin-conjugating E2 enzymes or UBE2s. In the present paper, we report a thorough screening of interactions between the TRIM and UBE2 families. We found a general preference of the TRIM proteins for the D and E classes of UBE2 enzymes, but we also revealed very specific interactions between TRIM9 and UBE2G2, and TRIM32 and UBE2V1/2. Furthermore, we demonstrated that the TRIM E3 activity is only manifest with the UBE2 with which they interact. For most specific interactions, we could also observe subcellular co-localization of the TRIM involved and its cognate UBE2 enzyme, suggesting that the specific selection of TRIM-UBE2 pairs has physiological relevance. Our findings represent the basis for future studies on the specific reactions catalysed by the TRIM E3 ligases to determine the fate of their targets.

The SUMO protease SENP6 is a direct regulator of PML nuclear bodies.

Promyelocytic leukemia protein (PML) is the core component of PML-nuclear bodies (PML NBs). The small ubiquitin-like modifier (SUMO) system (and, in particular, SUMOylation of PML) is a critical component in the formation and regulation of PML NBs. SUMO protease SENP6 has been shown previously to be specific for SUMO-2/3-modified substrates and shows preference for SUMO polymers. Here, we further investigate the substrate specificity of SENP6 and show that it is also capable of cleaving mixed chains of SUMO-1 and SUMO-2/3. Depletion of SENP6 results in accumulation of endogenous SUMO-2/3 and SUMO-1 conjugates, and immunofluorescence analysis shows accumulation of SUMO and PML in an increased number of PML NBs. Although SENP6 depletion drastically increases the size of PML NBs, the organizational structure of the body is not affected. Mutation of the catalytic cysteine of SENP6 results in its accumulation in PML NBs, and biochemical analysis indicates that SUMO-modified PML is a substrate of SENP6.

SUMOylation of the GTPase Rac1 is required for optimal cell migration.

The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1-GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.

Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies.

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.

Characterization of SENP7, a SUMO-2/3-specific isopeptidase.

The modification of proteins by SUMO (small ubiquitin-related modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SENPs [SUMO-1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidases]. To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (Ran GTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. in vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNA-mediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

Iso-seco-tanapartholides: Isolation, Synthesis and Biological Evaluation.

The isolation, identification and total synthesis of two plant-derived inhibitors of the NF-κB signaling pathway from the iso-seco-tanapartholide family of natural products is described. A key step in the efficient reaction sequence is a late-stage oxidative cleavage reaction that was carried out in the absence of protecting groups to give the natural products directly. A detailed comparison of the synthetic material with samples of the natural products proved informative. Biological studies on synthetic material confirmed that these compounds act late in the NF-κB signaling pathway. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009).