Ubiquitin-like proteins

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.

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.

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
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.

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.

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.

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