Dynamic Control of Selectivity in the Ubiquitination Pathway Revealed by an ASP to GLU Substitution in an Intra-Molecular Salt-Bridge Network
Sjoerd J. L. van Wijk
Adrien S. J. Melquiond
Sjoerd J. de Vries
H. Th. Marc Timmers
Alexandre M. J. J. Bonvin
- Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity.
Wie sekundäre Pflanzeninhaltsstoffe uns vor Krankheiten schützen : von molekularen Wirkmechanismen zu neuen Medikamenten
Jürgen M. Stein
- Wirkungen von Heilpflanzen, Gewürzen, Tees und Lebensmitteln werden in der Naturheilkunde seit der Antike genutzt. Pharmakologisch wirksam sind in der Regel nur die sekundären Pflanzeninhaltsstoffe. Diese in den oft aus vielen Bestandteilen zusammengesetzten Naturstoffen aufzuspüren und ihren molekularbiologischen Wirkungsmechanismus im Körper aufzuklären, ist das Ziel eines Forschungsnetzwerks am Frankfurter ZAFES (Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit). So konnten Pharmazeuten und Kliniker gemeinsam herausfinden, wie ein Bestandteil des Rotweins, das Resveratrol, vor Darmkrebs schützt. Die Inhaltsstoffe von Salbei und Rosmarin bieten vielversprechende Ausgangspunkte für neue Medikamente gegen Altersdiabetes. Weihrauch, Myrte und Johanniskraut enthalten Wirkstoffe, die Schlüsselenzyme für Entzündungsreaktionen – etwa bei rheumatischen Beschwerden – hemmen.
Hypoxic transcription gene profiles under the modulation of nitric oxide in nuclear run on-microarray and proteomics
Emeka I. Igwe
- Background: Microarray analysis still remains a powerful tool to identify new components of the transcriptosome and it has helped to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns, as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis. Result: We identified 188 genes that were significantly regulated by hypoxia, 81 genes were affected by nitric oxide, and 292 genes were induced by the co-treatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to hypoxia and/or nitric oxide treatments, but with different levels of expression. We observed that 166 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia regulated targets by NO. Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we increased the sensitivity of mRNA analysis and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signalling.
T-cell-specific peroxisome proliferator-activated receptor gamma depletion inhibits T-cell apoptosis and improves survival of septic mice via an IL-2-dependent mechanism
Martina V. Schmidt
Andreas von Knethen
- Poster presentation: from Sepsis 2009 Amsterdam, The Netherlands. 11-14 November 2009 Introduction Immune paralysis with massive T-cell apoptosis is a central pathogenic event during sepsis and correlates with septic patient mortality. Previous observations implied a crucial role of peroxisome proliferator-activated receptor gamma (PPARgamma) during T-cell apoptosis. Methods To elucidate mechanisms of PPARgamma-induced T-cell depletion, we used an endotoxin model as well as the caecal ligation and puncture sepsis model to imitate septic conditions in wild-type versus conditional PPARgamma knockout (KO) mice. Results PPARgamma KO mice showed a marked survival advantage compared with control mice. Their T cells were substantially protected against sepsis-induced death and showed a significantly higher expression of the pro-survival factor IL-2. Since PPARgamma is described to repress nuclear factor of activated T cells (NFAT) transactivation and concomitant IL-2 expression, we propose inhibition of NFAT as the underlying mechanism allowing T-cell apoptosis. Corroborating our hypothesis, we observed up-regulation of the pro-apoptotic protein BIM and downregulation of the anti-apoptotic protein Bcl-2 in control mice, which are downstream effector proteins of IL-2 receptor signaling. Application of a neutralizing anti-IL-2 antibody reversed the pro-survival effect of PPARgamma-deficient T cells and confirmed IL-2-dependent apoptosis during sepsis. Conclusion Apparently antagonizing PPARgamma in T cells might improve their survival during sepsis, which concomitantly enhances defence mechanisms and possibly provokes an increased survival of septic patients.
5-Lipoxygenase: underappreciated role of a pro-inflammatory enzyme in tumorigenesis
Astrid Stefanie Fischer
Svenja Dorothea Steinbrink
Thorsten Jürgen Maier
- Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
The ataxia (axJ) mutation causes abnormal GABAA receptor turnover in mice
Oliver B. Waidmann
Scott M. Wilson
- Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated axJ gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. AxJ mouse mutants, characterized by cerebellar ataxia, display both increased GABAA receptor (GABAAR) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABAAR alpha 1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABAAR turnover at cerebellar synapses contributes to axJ-mediated behavioural impairment.
Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment
Malcolm A. S. Moore
- Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.
IAPs as E3 ligases of Rac1 : shaping the move
Tripat Kaur Oberoi-Khanuja
- Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination–dependent inactivation of Rac1 signaling.
Ordnung der Fachbereiche Medizin, Biowissenschaften, Biochemie, Chemie und Pharmazie sowie Psychologie und Sportwissenschaften der Johann Wolfgang Goethe[-Universität] für den Masterstudiengang Interdisciplinary Neuroscience mit dem Abschluss "Master of Science" (M. Sc.) vom 30. März 2009 in der Fassung vom 22. April 2009, zuletzt geändert am 16. April 2012 : genehmigt vom Präsidium der Johann Wolfgang Goethe-Universität Frankfurt a. M. am 27. April 2010 ; hier: Änderungen vom 08.11.2012 ; genehmigt vom Präsidium der Johann Wolfgang Goethe-Universität Frankfurt a. M. am 18. Dezember 2012
Ordnung der Fachbereiche Medizin, Biowissenschaften, Biochemie, Chemie und Pharmazie sowie Psychologie und Sportwissenschaften der Johann Wolfgang Goethe-Universität für den Masterstudiengang Interdisciplinary Neuroscience mit dem Abschluss "Master of Science" (M.Sc.) vom 30. März 2009 in der Fassung vom 22. April 2009 : genehmigt durch das Präsidium am 11. Oktober 2011 ; hier: Änderung der Wahlpflichtmodule vom 16. April 2012 ; genehmigt durch das Präsidium am 11. September 2012