Ubiquitin-like proteins

The elimination of aberrant inclusions is regarded as a therapeutic approach in neurodegeneration. In amyotrophic lateral sclerosis (ALS), mutations in proteins found within cytoplasmic condensates called stress granules (SGs) are linked to the formation of pathological SGs, aberrant protein inclusions, and neuronal toxicity. We found that inhibition of NEDP1, the enzyme that processes/deconjugates the ubiquitin-like molecule NEDD8, promotes the disassembly of physiological and pathological SGs. Reduction in poly(ADP-ribose) polymerase1 activity through hyper-NEDDylation is a key mechanism for the observed phenotype. These effects are related to improved cell survival in human cells, and in C. elegans, nedp1 deletion ameliorates ALS phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, correlated to the disassembly of pathological SGs.

TRIM5α is a cytoplasmic restriction factor that blocks post-entry retroviral infection. Evidence suggests that its antiviral activity can be regulated by SUMO, but how this is achieved remains unknown. Here, we show that TRIM5α forms a complex with RanGAP1, Ubc9, and RanBP2 at the nuclear pore, and that RanBP2 E3 SUMO ligase promotes the SUMOylation of endogenous TRIM5α in the cytoplasm. Loss of RanBP2 blocked SUMOylation of TRIM5α, altered its localization in primary cells, and suppressed the antiviral activity of both rhesus and human orthologs. In cells, human TRIM5α is modified on K84 within a predicted phosphorylated SUMOylation motif (pSUM) and not on K10 as found in vitro. Non-modified TRIM5α lacked antiviral activity, indicating that only SUMOylated TRIM5α acts as a restriction factor. This work illustrates the importance of the nuclear pore in intrinsic antiviral immunity, acting as a hub where virus, SUMO machinery, and restriction factors can meet.

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin−RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8-and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas sitespecific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

In the version of this article initially published, in the caption for Extended Data Fig. 4i, Extended Data Fig. 1j was incorrectly cited as "Supplementary fig. 1j". The error has been corrected in the HTML and PDF versions of the article. In the supplementary information originally posted for this article, Supplementary Table 1 did not accurately reflect the oligonucleotide sequences used in this study. The last three sequences in the table, "polyA LNA CO", "polyA PO" and "polyA EO" were not used in this study. These have been replaced with the sequences that were used: "TERC LNA CO", TERC PO" and "TERC EO". The errors have been corrected online.

In order to maintain cellular homeostasis, the activity of several thousand proteins needs to be carefully orchestrated in a spatio-temporal manner, and multiple layers of regulation exist to ensure that protein function is properly carried out. Critical regulatory mechanisms include trafficking of proteins to specific subcellular compartments and post-translational modifications (PTMs), such as ubiquitylation or sumoylation. These mechanisms often operate in a coordinated manner to determine the levels, localization and activity of a protein. In particular, the functional relationships between active nucleocytoplasmic transport and the ubiquitin/SUMO modification pathways are becoming progressively elucidated. In this review, we focus on the interplay between these two processes, and its importance in cancer. We begin by presenting an overview of the processes of protein ubiquitylation and sumoylation, with an emphasis on the cross-regulation that exists between the nuclear transport machinery and the ubiquitin/SUMO modification enzymes. Next, we focus on the regulation of the important tumor proteins p53 and PTEN as examples to illustrate how these processes cooperate to modulate the activity of cancer-related proteins. Importantly, novel drugs are being developed that target proteins playing a role in nuclear transport (e.g. the nuclear export receptor CRM1) and ubiquitin/SUMO modifications (e.g. ubiquitin E3 ligases and deubiquitinases). A deeper understanding of the interplay between these processes may facilitate in the future the rational combination of these novel agents.

TRIM5α is a cytoplasmic restriction factor that blocks post-entry retroviral infection. Evidence suggests that its antiviral activity can be regulated by SUMO, but how this is achieved remains unknown. Here, we show that TRIM5α forms a complex with RanGAP1, Ubc9, and RanBP2 at the nuclear pore, and that RanBP2 E3 SUMO ligase promotes the SUMOylation of endogenous TRIM5α in the cytoplasm. Loss of RanBP2 blocked SUMOylation of TRIM5α, altered its localization in primary cells, and suppressed the antiviral activity of both rhesus and human orthologs. In cells, human TRIM5α is modified on K84 within a predicted phosphorylated SUMOylation motif (pSUM) and not on K10 as found in vitro. Non-modified TRIM5α lacked antiviral activity, indicating that only SUMOylated TRIM5α acts as a restriction factor. This work illustrates the importance of the nuclear pore in intrinsic antiviral immunity, acting as a hub where virus, SUMO machinery, and restriction factors can meet.

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative disorders that cause deterioration of cognitive functions. Recent studies suggested that the accumulation of inflammatory molecules and impaired protein degradation mechanisms might both play a critical role in the progression of AD. Autophagy is a major protein degradation pathway that can be controlled by several HECT-E ligases, which then regulates the expression of inflammatory molecules. E ubiquitin ligases are known to be upregulated in several neurodegenerative diseases. Here, we studied the expressional change of HECT-E ligase using M on autophagy and inflammasome pathways in the context of AD pathogenesis. Our results demonstrated that the M treatment reversed the working memory deficits in xTg-AD mice when examined with the T-maze and reversal learning with the Morris water maze. Additionally, the electrophysiology recordings indicated that M treatment enhanced the long-term potentiation in the hippocampus of xTg-AD mice. Together with the improved memory performance, the expression levels of the NLRP inflammasome protein were decreased. On the other hand, autophagy-related molecules were increased in the hippocampus of xTg-AD mice. Furthermore, the protein docking analysis indicated that the binding a nity of M to the WWP and NEDD E ligases was the highest among the HECT family members. The western blot analysis also confirmed the decreased expression level of NEDD protein in the M-treated xTg-AD mice. Overall, our results demonstrate that the modulation of HECT-E ligase expression level can be used as a strategy to treat early memory deficits in AD by decreasing NLRP inflammasome molecules and increasing the autophagy pathway.

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative disorders that cause deterioration of cognitive functions. Recent studies suggested that the accumulation of inflammatory molecules and impaired protein degradation mechanisms might both play a critical role in the progression of AD. Autophagy is a major protein degradation pathway that can be controlled by several HECT-E ligases, which then regulates the expression of inflammatory molecules. E ubiquitin ligases are known to be upregulated in several neurodegenerative diseases. Here, we studied the expressional change of HECT-E ligase using M on autophagy and inflammasome pathways in the context of AD pathogenesis. Our results demonstrated that the M treatment reversed the working memory deficits in xTg-AD mice when examined with the T-maze and reversal learning with the Morris water maze. Additionally, the electrophysiology recordings indicated that M treatment enhanced the long-term potentiation in the hippocampus of xTg-AD mice. Together with the improved memory performance, the expression levels of the NLRP inflammasome protein were decreased. On the other hand, autophagy-related molecules were increased in the hippocampus of xTg-AD mice. Furthermore, the protein docking analysis indicated that the binding a nity of M to the WWP and NEDD E ligases was the highest among the HECT family members. The western blot analysis also confirmed the decreased expression level of NEDD protein in the M-treated xTg-AD mice. Overall, our results demonstrate that the modulation of HECT-E ligase expression level can be used as a strategy to treat early memory deficits in AD by decreasing NLRP inflammasome molecules and increasing the autophagy pathway.

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin−RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8-and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas sitespecific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

Alzheimer's disease (AD) is one of the most common progressive neurodegenerative disorders that cause deterioration of cognitive functions. Recent studies suggested that the accumulation of inflammatory molecules and impaired protein degradation mechanisms might both play a critical role in the progression of AD. Autophagy is a major protein degradation pathway that can be controlled by several HECT-E ligases, which then regulates the expression of inflammatory molecules. E ubiquitin ligases are known to be upregulated in several neurodegenerative diseases. Here, we studied the expressional change of HECT-E ligase using M on autophagy and inflammasome pathways in the context of AD pathogenesis. Our results demonstrated that the M treatment reversed the working memory deficits in xTg-AD mice when examined with the T-maze and reversal learning with the Morris water maze. Additionally, the electrophysiology recordings indicated that M treatment enhanced the long-term potentiation in the hippocampus of xTg-AD mice. Together with the improved memory performance, the expression levels of the NLRP inflammasome protein were decreased. On the other hand, autophagy-related molecules were increased in the hippocampus of xTg-AD mice. Furthermore, the protein docking analysis indicated that the binding a nity of M to the WWP and NEDD E ligases was the highest among the HECT family members. The western blot analysis also confirmed the decreased expression level of NEDD protein in the M-treated xTg-AD mice. Overall, our results demonstrate that the modulation of HECT-E ligase expression level can be used as a strategy to treat early memory deficits in AD by decreasing NLRP inflammasome molecules and increasing the autophagy pathway.

Background Mitochondrial diseases represent one of the most common groups of genetic diseases. With a prevalence greater than 1 in 5000 adults, such diseases still lack effective treatment. Current therapies are purely palliative and, in most cases, insufficient. Novel approaches to compensate and, if possible, revert mitochondrial dysfunction must be developed. Results In this study, we tackled the issue using as a model fibroblasts from a patient bearing a mutation in the GFM1 gene, which is involved in mitochondrial protein synthesis. Mutant GFM1 fibroblasts could not survive in galactose restrictive medium for more than 3 days, making them the perfect screening platform to test several compounds. Tetracycline enabled mutant GFM1 fibroblasts survival under nutritional stress. Here we demonstrate that tetracycline upregulates the mitochondrial Unfolded Protein Response (UPRmt), a compensatory pathway regulating mitochondrial proteostasis. We additionally report that activation of ...

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of homologous recombination. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin...

The interrelationship between endogenous microbiota, the immune system, and tissue regeneration is an area of intense research due to its potential therapeutic applications. We investigated this relationship in Schmidtea mediterranea, a model organism capable of regenerating any and all of its adult tissues. Microbiome characterization revealed a high Bacteroidetes to Proteobacteria ratio in healthy animals. Perturbations eliciting an expansion of Proteobacteria coincided with ectopic lesions and tissue degeneration. The culture of these bacteria yielded a strain of Pseudomonas capable of inducing progressive tissue degeneration. RNAi screening uncovered a TAK1 innate immune signaling module underlying compromised tissue homeostasis and regeneration during infection. TAK1/MKK/p38 signaling mediated opposing regulation of apoptosis during infection versus normal tissue regeneration. Given the complex role of inflammation in either hindering or supporting reparative wound healing and regeneration, this invertebrate model provides a basis for dissecting the duality of evolutionarily conserved inflammatory signaling in complex, multi-organ adult tissue regeneration.

Upon exposure to harmful microorganisms, hosts engage in protective molecular and behavioral immune responses, both of which are ultimately regulated by the nervous system. Using the nematode Caenorhabditis elegans, we show that ingestion of Enterococcus faecalis leads to a fast pathogen avoidance behavior that results in aversive learning. We have identified multiple sensory mechanisms involved in the regulation of avoidance of E. faecalis. The G-protein coupled receptor NPR-1-dependent oxygen-sensing pathway opposes this avoidance behavior, while an ASE neuron-dependent pathway and an AWB and AWC neuron-dependent pathway are directly required for avoidance. Colonization of the anterior part of the intestine by E. faecalis leads to AWB and AWC mediated olfactory aversive learning. Finally, two transient receptor potential melastatin (TRPM) channels, GON-2 and GTL-2, mediate this newly described rapid pathogen avoidance. These results suggest a mechanism by which TRPM channels may s...

Posttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from~130 pN to~170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N-and C-termini) increased upon SBM binding from~1 N/m to~3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.

Post-translational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes during which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact non-covalently with SUMOs and induce conformational changes. In order to determine the effect of SBMs on the mechanical properties of SUMO1 (first member of the human SUMO family), we have performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400nm/s) increased from ~130 pN to ~ 170 pN upon binding to SBMs indicating mechanical stabilization upon complexation. Pulling speed dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding ...

Innate immunity is an evolutionary ancient defence strategy that serves to eliminate infectious agents while maintaining host health. It involves a complex network of sensors, signaling proteins and immune effectors that detect the danger, then relay and execute the immune programme. Post-translational modifications relying on conserved ubiquitin and ubiquitin-like proteins are an integral part of the system. Studies using invertebrate models of infection, such as the nematode Caenorhabditis elegans, have greatly contributed to our understanding of how ubiquitin-related processes act in immune sensing, regulate immune signaling pathways, and participate to host defence responses. This review highlights the interest of working with a genetically tractable model organism and illustrates how C. elegans has been used to identify ubiquitin-dependent immune mechanisms, discover novel ubiquitin-based resistance strategies that mediate pathogen clearance, and unravel the role of ubiquitin-related processes in tolerance, preserving host fitness during pathogen attack. Special emphasis is placed on processes that are conserved in mammals.

Ubiquitination of G protein-coupled receptors (GPCRs) is an important dynamic posttranslational modification that has been linked to the intracellular trafficking of internalized GPCRs to lysosomes. Ubiquitination of GPCRs is mediated by specific E3 ubiquitin ligases that are scaffolded by the adaptor proteins called β-arrestins. Traditionally, detection of GPCR ubiquitination is achieved by using ubiquitin antibodies to Western blot immunoprecipitates of detergent-solubilized GPCRs expressed in heterologous cells. However, studies have also shown that bioluminescence resonance energy transfer (BRET)-based techniques can reveal ubiquitination of GPCRs in intact cells and in real time. This chapter describes a step-by-step protocol to evaluate ubiquitination of GPCRs using the BRET methodology.

We present a novel approach to the investigation of rapid (>2 s À1 ) NH exchange rates in proteins, based on residue-specific diffusion measurements. 1 H, 15 N-DOSY-HSQC spectra are recorded in order to observe resolved amide proton signals for most residues of the protein. Human ubiquitin was used to demonstrate the proposed method. Exchange rates are derived directly from the decay data of the diffusion experiment by applying a model deduced from the assumption of a two-site exchange with water and the ''pure'' diffusion coefficients of water and protein. The ''pure'' diffusion coefficient of the protein is determined in an experiment with selective excitation of the amide protons in order to suppress the influence of magnetization transfer from water to amide protons on the decay data. For rapidly exchanging residues a comparison of our results with the exchange rates obtained in a MEXICO experiment showed good agreement. Molecular dynamics (MD) and quantum mechanical calculations were performed to find molecular parameters correlating with the exchangeability of the NH protons. The RMS fluctuations of the amide protons, obtained from the MD simulations, together with the NH coupling constants provide a bilinear model which shows a good correlation with the experimental NH exchange rates.

A rare γ-pyranopyrazole skeleton: design, one-pot synthesis and computational study †
Drawing upon a consecutive amide coupling and intramolecular cyclisation pathway, a one-pot, straightforward synthetic route has been developed for a range of pyrazole fused γ-pyrone derivatives. The reaction mechanism proposed for the chemoselective formation of γ-pyranopyrazole is furthermore fully supported by experimental and computational studies.

Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the neddylation defect of yeast dcn1⌬ cells. DCNL3 down-regulation by RNAi decreases Cul neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.

Nóbelsverðlaunahafarnir í efnafræði 2004, Aaron Ciechanover (f. 1947), Avram Herskho (f. 1937) og Irwin Rose (f. 1926) uppgötvuðu í sameiningu ubiquitin-miðlað próteinniðurbrot, ferli þar sem lífhvatakerfi tengir fjölmargar sameindir ubiquitins við prótein sem fruman þarf að losa sig við. Ákvörðun um veitingu Nóbelsverðlaunanna 2004 byggði einkum á rannsóknum sem þeir framkvæmdu á fyrri hluta níunda áratugarins. Aaron Ciechanover og Avram Herskho hafa lengst af starfað á Institute of Technology Haifa, Israel. Einnig störfuðu þeir báðir sem gestavísindamenn (þá var Ciechanover enn doktorsnemandi) á rannsóknastofu Irwin Rose, Institute for Cancer Research, Fox Chase Center í Philadelphiu Bandaríkjunum. Eftir doktorsprófið 1981 færði Ciechanover sig yfir til Massachusetts Institute of Technology (MIT) í Boston í Bandaríkjunum. Síðustu árin hefur Irwin Rose starfað við Kaliforníusháskólann í Irvine.

Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the neddylation defect of yeast dcn1⌬ cells. DCNL3 down-regulation by RNAi decreases Cul neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K*SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (ΨKXE), and functions only in the context of an ␣-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

Posttranslational modification with small ubiquitinrelated modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9$SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

O.Kirsh and J.-S.Seeler contributed equally to this work repressor (MITR) as well as HDAC1 and -6 are similarly SUMO modi®ed, indicating that sumoylation may be an important regulatory mechanism for the control of transcriptional repression mediated by both class I and II HDACs.

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K*SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (ΨKXE), and functions only in the context of an ␣-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

Post-translational modification with the ubiquitin-related protein SUMO1 requires the E1 enzyme Aos1–Uba2 and the E2 enzyme
Ubc9. Distinct E3 ligases strongly enhance modification of specific targets. The SUMO E3 ligase RanBP2 (also known as Nup358)
has no obvious similarity to RING- or HECT-type enzymes. Here we show that RanBP2’s 30-kDa catalytic fragment is a largely
unstructured protein. Despite two distinct but partially overlapping 79-residue catalytic domains, one of which is sufficient for
maximal activity, RanBP2 binds to Ubc9 in a 1:1 stoichiometry. The identification of nine RanBP2 and three Ubc9 side chains that
are important for RanBP2-dependent SUMOylation indicates largely hydrophobic interactions. These properties distinguish
RanBP2 from all other known E3 ligases, and we speculate that RanBP2 exerts its catalytic effect by altering Ubc9’s properties
rather than by mediating target interactions.

Aos1/Uba2) and a single E2 conjugating enzyme (Ubc9) have been identified in yeast and higher eukaryotes. In vitro, these are sufficient to modify a number of SUMO1 82152 Martinsried Mü ller et al., 2001). However, recently several SUMO Germany E3-like factors were identified in yeast and mammalian 2 Unité de Recombinaison et Expression Gé né tique cells (Johnson and Gupta, 2001; Kahyo et al., 2001; INSERM U163 Sachdev et al., 2001; Takahashi et al., 2001). These pro-Institut Pasteur teins are different members of one family, the protein 28 rue du Dr. Roux inhibitors of activated STAT (PIAS

Posttranslational modification with the small
ubiquitin-related modifier SUMO depends on the
sequential activities of E1, E2, and E3 enzymes. While
regulation by E3 ligases and SUMO proteases is well
understood, current knowledge of E2 regulation is
very limited. Here, we describe modification of the
budding yeast E2 enzyme Ubc9 by sumoylation
(Ubc9*SUMO). Although less than 1% of Ubc9 is
sumoylated at Lys153 at steady state, a sumoylation-
deficient mutant showed significantly reduced
meiotic SUMO conjugates and abrogates synaptonemal
complex formation. Biochemical analysis
revealed that Ubc9*SUMO is severely impaired in
its classical activity but promoted SUMO chain
assembly in the presence of Ubc9. Ubc9*SUMO
cooperates with charged Ubc9 (Ubc9
SUMO) by
noncovalent backside SUMO binding and by positioning
the donor SUMO for optimal transfer. Thus,
sumoylation of Ubc9 converts an active enzyme
into a cofactor and reveals a mechanism for E2 regulation
that orchestrates catalytic (Ubc9
SUMO) and
noncatalytic (Ubc9*SUMO) functions of Ubc9.

Posttranslational modification with small ubiquitinrelated
modifier, SUMO, is a widespread mechanism
for rapid and reversible changes in protein function.
Considering the large number of known targets, the
number of enzymes involved in modification seems
surprisingly low: a single E1, a single E2, and a few
distinct E3 ligases. Here we show that autosumoylation
of the mammalian E2-conjugating enzyme Ubc9
at Lys14 regulates target discrimination. While not
altering its activity toward HDAC4, E2-25K, PML, or
TDG, sumoylation of Ubc9 impairs its activity on
RanGAP1 and strongly activates sumoylation of the
transcriptional regulator Sp100. Enhancement depends
on a SUMO-interacting motif (SIM) in Sp100
that creates an additional interface with the SUMO
conjugated to the E2, a mechanism distinct from
Ubc9SUMO thioester recruitment. The crystal
structure of sumoylated Ubc9 demonstrates how the
newly created binding interface can provide a gain in
affinity otherwise provided by E3 ligases.

Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the neddylation defect of yeast dcn1⌬ cells. DCNL3 down-regulation by RNAi decreases Cul neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.

The present work provides a new approach to evolve ligand structures which represent possible drug to be docked to the active site of the target protein. The structure is represented as a tree where each non-empty node represents a functional group. It is assumed that the active site configuration of the target protein is known with position of the essential residues. In this paper the interaction energy of the ligands with the protein target is minimized. Moreover, the size of the tree is difficult to obtain and it will be different for different active sites. To overcome the difficulty, a variable tree size configuration is used for designing ligands. The optimization is done using a quantum discrete PSO. The result using fixed length and variable length configuration are compared.