Simin Rahighi

Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB Activation

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Unusual antibacterial property of mesoporous titania films: drastic improvement by controlling surface area and crystallinity.

One of the most urgent requirements of human life in the 21 century is development of new antibacterial materials and sterilization technologies that can improve human health. Until now, the most commonly used antibacterial agents are based on chlorine, chlorine dioxide, and organic biocide compounds. These agents are extremely toxic for humans and their residues are also not environmentally friendly. Therefore, it is very important to develop antibacterial biocompatible materials. Titanium dioxide (titania, TiO2) materials in the anatase form have attracted great interest as a new antibacterial material. Titania can work well under ultraviolet (UV) light owing to its photo-semiconductor properties. Currently, this property has been widely utilized for various applications such as water treatment, air and environmental purification, hazardous waste remediation, and deactivation of bacteria. Commercial products with titania (e.g., self-cleaning glasses and anti-fogging coatings) are well known all over the world. To date, various titania-based nanostructures including nanorods and nanoparticles have been reported. To further enhance the photocatalytic performance, many efforts have been made for doping various metal/semiconductor elements into titania materials. In this system, the electrons accumulated on the metal and holes remained on the photocatalyst surface. Therefore, a significant reduction in the recombination rate is realized owing to better charge separation between the electrons and holes. Therefore, the titania-based composites with metal/semiconductor elements can enhance the overall photocatalytic efficiency and the damage of microorganisms of the cell. In this Communication, we focused on a further simple and low-cost synthetic method and synthesized mesoporous titania films by utilizing bottom-up nanotechnology with surfactant assembly. Mesoporous materials with extremely high surface area should be good candidates for the next generation of antibacterial materials. Compared with the traditional titania materials mentioned above, the high surface area originated from mesoporous networks can provide a higher amount of hydroxyl radicals (·OH) which can increase the photoactivity. In the past few years, special attention has been paid to the synthesis of mesoporous titania powders as effective photocatalysts including an antibacterial application. However, the mesoporous titania in the powder form has some disadvantages. The powders which are not fixed on substrates are washed out easily by external treatments and then the released particles themselves may pollute the environment. Also, nanosized powders generally cause serious problems to human health. Therefore, the mesoporous titania films reported here are more applicable [a] H. Oveisi, X. Jiang, Dr. Y. Nemoto, Prof. Dr. Y. Yamauchi World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS) 1-1 Namiki, Tsukuba, Ibaraki, 305-0044 (Japan) Fax: (+81)29-860-4706 E-mail : Yamauchi.Yusuke@nims.go.jp [b] Dr. S. Rahighi, Prof. Dr. S. Wakatsuki Structural Biology Research Center High Energy Accelerator Research Organization (KEK) 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan) [c] H. Oveisi, Prof. Dr. A. Beitollahi Center of Excellence in Advanced Materials and Processing Department of Metallurgy and Materials Engineering Iran University of Science and Technology (IUST) Narmak, Tehran 16844 (Iran) [d] X. Jiang, Prof. Dr. Y. Yamauchi Faculty of Science and Engineering Waseda University 3-4-1 Okubo, Shinjuku, Tokyo 169-8555 (Japan) [e] Prof. Dr. Y. Yamauchi Precursory Research for Embryonic Science and Technology (PRESTO) Japan Science and Technology Agency (JST) 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan). [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201000351.

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

ABSTRACT The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.

Crystal Structure of a PCP/Sfp Complex Reveals the Structural Basis for Carrier Protein Posttranslational Modification

Phosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier proteins second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life.

Selectivity of the ubiquitin-binding modules

Ubiquitin‐binding modules are constituents of cellular proteins that mediate the effects of ubiquitylation by making transient, non‐covalent interactions with ubiquitin molecules. While some ubiquitin‐binding modules bind single ubiquitin moieties, others are selective for specific ubiquitin chains of different linkage types and lengths. In recent years, functions of ubiquitin chains that are polymerized through their Lys or N‐terminal Met (i.e. linear chains) residues have been linked to a variety of cellular processes. Selectivity of ubiquitin‐binding modules for different ubiquitin chain types appears as a key to the distinct regulatory consequences during protein quality control pathways, receptor endocytosis, gene transcription, signaling via the NF‐κB pathway, and autophagy.

Selective Binding of AIRAPL Tandem UIMs to Lys48-Linked Tri-Ubiquitin Chains

Lys48-linked ubiquitin chains act as the main targeting signals for protein degradation by the proteasome. Here we report selective binding of AIRAPL, a protein that associates with the proteasome upon exposure to arsenite, to Lys48-linked tri-ubiquitin chains. AIRAPL comprises two ubiquitin-interacting motifs in tandem (tUIMs) that are linked through a flexible inter-UIM region. In the complex crystal structure UIM1 binds the proximal ubiquitin, whereas UIM2 (the double-sided UIM) binds non-symmetrically to the middle and distal ubiquitin moieties on either side of the helix. Specificity of AIRAPL for Lys48-linked ubiquitin chains is determined by UIM2, and the flexible inter-UIM linker increases avidity by placing the two UIMs in an orientation that facilitates binding of the third ubiquitin to UIM1. Unlike middle and proximal ubiquitins, distal ubiquitin binds UIM2 through a novel surface, which leaves the Ile44 hydrophobic patch accessible for binding to the proteasomal ubiquitin receptors.

Selective Binding of Linear Ubiquitin Chains to NEMO in NF-kappaB Activation

Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) depends on multiple ubiquitination and phosphorylation signals. For example, an acute stimulation of cells with variety of cytokines leads to Lys63-linked ubiquitin chain conjugation on the receptor-associated complexes to activate the TAK1 kinase. In addition, function of the linear ubiquitin chain assembly (LUBAC) ligase is required for the activation of IkappaB kinase complex (IKK), which in turn phosphorylates IkappaB-alpha and causes its proteasomal degradation via Lys48-linked ubiquitin-chain conjugation. The directionality and the specificity in the NF-kappaB pathway are accomplished by the specific ubiquitin receptors that are able to recognize specific ubiquitin signals. We have provided structural and biochemical evidences for a selective binding of the NEMO–UBAN (Ubiquitin Binding in ABIN and NEMO) motif to linear (head-to-tail) ubiquitin chains. The NEMO–UBAN forms a parallel coiled-coil dimer, which binds to two linear diubiquitin molecules perpendicularly positioned on each side of the NEMO dimer. Residues of NEMO involved in binding to linear ubiquitin chains are essential for NF-kappaB activation by TNF-alpha and other agonists and are found mutated in human disease characterized by ectodermal dysplasia with immunodeficiency.

Linear ubiquitin chain‐binding domains

Ubiquitin modification (ubiquitination) of target proteins can vary with respect to chain lengths, linkage type, and chain forms, such as homologous, mixed, and branched ubiquitin chains. Thus, ubiquitination can generate multiple unique surfaces on a target protein substrate. Ubiquitin‐binding domains (UBDs) recognize ubiquitinated substrates, by specifically binding to these unique surfaces, modulate the formation of cellular signaling complexes and regulate downstream signaling cascades. Among the eight different homotypic chain types, Met1‐linked (also termed linear) chains are the only chains in which linkage occurs on a non‐Lys residue of ubiquitin. Linear ubiquitin chains have been implicated in immune responses, cell death and autophagy, and several UBDs ‐ specific for linear ubiquitin chains ‐ have been identified. In this review, we describe the main principles of ubiquitin recognition by UBDs, focusing on linear ubiquitin chains and their roles in biology.

Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.Glucose-6-phosphate dehydrogenase (G6PD) deficiency provides insufficient protection from oxidative stress, contributing to diverse human pathologies. Here, the authors identify a small molecule that increases the activity and/or stability of mutant G6PD and show that it reduces oxidative stress in zebrafish and hemolysis in isolated human erythrocytes.

Conformational flexibility and rotation of the RING domain in activation of cullin–RING ligases

The RING protein RBX-1 is implicated in both NEDDylation and ubiquitylation reactions. In this issue, new structural analysis reveals how conformational flexibility of the RBX-1 linker allows for a marked reorientation of the CUL1–RBX1 complex to facilitate transfer of NEDD8 or ubiquitin by closing the gap between the E2 catalytic site and the substrate.