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News Article | May 10, 2017
Site: www.eurekalert.org

All bacteria have developed clever mechanisms for survival and propagation within host cells. Salmonella are a typical example: usually they hide in membrane-bound particles with only very few bacteria escaping to the cell's interior. Those escapees are extremely dangerous as they proliferate and spread at enormous speed. To stop such an invasion, cells have developed very effective defense strategies. An interdisciplinary team around Prof. Ivan Dikic (Institute of Biochemistry II) and Prof. Mike Heilemann (Institute of Physical and Theoretical Chemistry), both from Goethe University Frankfurt, now studied such a cellular defense mechanism by visualizing protein patterns at the near-molecular level. Upon bacterial invasion, cells react fast: They flag escaped bacteria with a small protein called ubiquitin, which is known to regulate numerous cellular processes. The attached flags contain chains of differently linked ubiquitin molecules, resulting in a secret code, which has so far only partially been decoded. Similar to mobile transmission towers, these ubiquitin chains relay specific signals from the surface of the bacteria into the cell. Employing super-resolution microscopy, the Frankfurt team now succeeded with visualizing different ubiquitin chains on the bacterial surface and analyzing their molecular organization in detail. They discovered that one chain type, so called linear chains, plays an essential role during a bacterial invasion. Linear ubiquitin chains trigger degradation of bacteria and kick off an inflammatory signaling cascade which results in restricting bacterial proliferation. In addition, the researchers identified the enzyme Otulin as an important regulator capable of limiting this reaction - a very important notion considering the fact that excessive inflammation is one of the major causes of tissue damage following bacterial infection. Signaling the cells' need for pathogen defense is just one important role of ubiquitin. The small protein is also involved in development and progression of inflammatory and neurodegenerative diseases as well as of cancer. Until now, however, very little is known about how small errors in the ubiquitin system contribute to these serious human diseases, and how the system can be targeted pharmaceutically. These new findings pave the way for many follow-up projects which may ultimately lead to novel therapeutic approaches. Very recently, Ivan Dikic obtained one of the prestigious ERC Advanced Grants of 2.5 M € in which he will investigate the role of ubiquitin in modulating the host-pathogen interaction in more detail. The work of the Frankfurt team is an excellent example for interdisciplinary collaboration and was enabled by funding of several large research networks, e.g. the Cluster of Excellence Macromolecular Complexes, the CRC 1177 on selective autophagy and the LOEWE ubiquitin network. The results are now published in the latest online issue of Nature Microbiology, back-to-back with complementary insights generated by colleagues in Cambridge (UK).


News Article | May 10, 2017
Site: phys.org

Salmonella within a human cell, surrounded by a coat of different ubiquitin chains. Purple represents linear ubiquitin chains, green all ubiquitin chains. Visualized by super-resolution microscopy (dSTORM). Credit: Mike Heilemann/Ivan Dikic With drug resistance being on the rise worldwide, bacterial infections pose one of the greatest global threats to human health. By deciphering the host-pathogen interaction on a molecular level, researchers hope to pave the way for new therapies. Studying the cell's reaction to Salmonella, scientists from Goethe University Frankfurt have now made a critical discovery to this respect. All bacteria have developed clever mechanisms for survival and propagation within host cells. Salmonella are a typical example: usually they hide in membrane-bound particles with only very few bacteria escaping to the cell's interior. Those escapees are extremely dangerous as they proliferate and spread at enormous speed. To stop such an invasion, cells have developed very effective defense strategies. An interdisciplinary team around Prof. Ivan Dikic (Institute of Biochemistry II) and Prof. Mike Heilemann (Institute of Physical and Theoretical Chemistry), both from Goethe University Frankfurt, now studied such a cellular defense mechanism by visualizing protein patterns at the near-molecular level. Upon bacterial invasion, cells react fast: They flag escaped bacteria with a small protein called ubiquitin, which is known to regulate numerous cellular processes. The attached flags contain chains of differently linked ubiquitin molecules, resulting in a secret code, which has so far only partially been decoded. Similar to mobile transmission towers, these ubiquitin chains relay specific signals from the surface of the bacteria into the cell. Employing super-resolution microscopy, the Frankfurt team now succeeded with visualizing different ubiquitin chains on the bacterial surface and analyzing their molecular organization in detail. They discovered that one chain type, so called linear chains, plays an essential role during a bacterial invasion. Linear ubiquitin chains trigger degradation of bacteria and kick off an inflammatory signaling cascade which results in restricting bacterial proliferation. In addition, the researchers identified the enzyme Otulin as an important regulator capable of limiting this reaction – a very important notion considering the fact that excessive inflammation is one of the major causes of tissue damage following bacterial infection. Signaling the cells' need for pathogen defense is just one important role of ubiquitin. The small protein is also involved in development and progression of inflammatory and neurodegenerative diseases as well as of cancer. Until now, however, very little is known about how small errors in the ubiquitin system contribute to these serious human diseases, and how the system can be targeted pharmaceutically. These new findings pave the way for many follow-up projects which may ultimately lead to novel therapeutic approaches. Very recently, Ivan Dikic obtained one of the prestigious ERC Advanced Grants of 2.5 M € in which he will investigate the role of ubiquitin in modulating the host-pathogen interaction in more detail. Explore further: Tracing down linear ubiquitination: New technology enables detailed analysis of target proteins


News Article | May 10, 2017
Site: www.biosciencetechnology.com

All bacteria have developed clever mechanisms for survival and propagation within host cells. Salmonella are a typical example: usually they hide in membrane-bound particles with only very few bacteria escaping to the cell's interior. Those escapees are extremely dangerous as they proliferate and spread at enormous speed. To stop such an invasion, cells have developed very effective defense strategies. An interdisciplinary team around Prof. Ivan Dikic (Institute of Biochemistry II) and Prof. Mike Heilemann (Institute of Physical and Theoretical Chemistry), both from Goethe University Frankfurt, now studied such a cellular defense mechanism by visualizing protein patterns at the near-molecular level. Upon bacterial invasion, cells react fast: They flag escaped bacteria with a small protein called ubiquitin, which is known to regulate numerous cellular processes. The attached flags contain chains of differently linked ubiquitin molecules, resulting in a secret code, which has so far only partially been decoded. Similar to mobile transmission towers, these ubiquitin chains relay specific signals from the surface of the bacteria into the cell. Employing super-resolution microscopy, the Frankfurt team now succeeded with visualizing different ubiquitin chains on the bacterial surface and analyzing their molecular organization in detail. They discovered that one chain type, so called linear chains, plays an essential role during a bacterial invasion. Linear ubiquitin chains trigger degradation of bacteria and kick off an inflammatory signaling cascade which results in restricting bacterial proliferation. In addition, the researchers identified the enzyme Otulin as an important regulator capable of limiting this reaction - a very important notion considering the fact that excessive inflammation is one of the major causes of tissue damage following bacterial infection. Signaling the cells' need for pathogen defense is just one important role of ubiquitin. The small protein is also involved in development and progression of inflammatory and neurodegenerative diseases as well as of cancer. Until now, however, very little is known about how small errors in the ubiquitin system contribute to these serious human diseases, and how the system can be targeted pharmaceutically. These new findings pave the way for many follow-up projects which may ultimately lead to novel therapeutic approaches. Very recently, Ivan Dikic obtained one of the prestigious ERC Advanced Grants of 2.5 M € in which he will investigate the role of ubiquitin in modulating the host-pathogen interaction in more detail. The work of the Frankfurt team is an excellent example for interdisciplinary collaboration and was enabled by funding of several large research networks, e.g. the Cluster of Excellence Macromolecular Complexes, the CRC 1177 on selective autophagy and the LOEWE ubiquitin network. The results are now published in the latest online issue of Nature Microbiology, back-to-back with complementary insights generated by colleagues in Cambridge (UK).


News Article | December 1, 2016
Site: www.eurekalert.org

The attachment of ubiquitin was long considered as giving the „kiss of death", labelling superfluous proteins for disposal within a cell. However, by now it has been well established that ubiquitin fulfils numerous additional duties in cellular signal transduction. A team of scientists under the lead of Ivan Dikic, Director of the Institute of Biochemistry II at Goethe University Frankfurt, has now discovered a novel mechanism of ubiquitination, by which Legionella bacteria can seize control over their host cells. Legionella causes deadly pneumonia in immunocompromised patients. According to the current understanding, the coordinated action of three enzymes is needed for attaching ubiquitin to other proteins. In April this year, U.S. scientists described an ubiquitination reaction that depends only on a single enzyme from Legionella bacteria. The Dikic team together with the group of Ivan Matic (Max Planck Institute for Biology of Ageing, Cologne, Germany) now elucidated the underlying molecular mechanism of its action and revealed a hitherto unknown type of chemical linkage between ubiquitin and target proteins. Their discovery breaks new ground in the field. Sagar Bhogaraju, researcher in the Dikic laboratory, comments: "Most exciting is of course the question if this unusual ubiquitination also occurs in human cells independently of bacterial infection and if there are similar, so far unknown enzymes in humans, which may have a profound influence on cellular processes." When studying the new mechanism in more detail, the Frankfurt scientists were very surprised to find that the Legionella enzyme does not only transfer ubiquitin onto target proteins, but also chemically manipulates the remaining pool of ubiquitin molecules. Modified ubiquitin almost completely inhibits the conventional ubiquitination system, thereby revealing a new role for this enzyme during Legionella infections. Several important cellular processes are affected by this shut-down of the ubiquitination system, which can also cause a rapid cell death. The Dikic team showed for example that modified ubiquitin prevents degradation of mitochondria (a process called mitophagy), affects transduction of inflammatory signals and constrains protein degradation. "Most likely, Legionella is not the only bacterium using this mechanism. We hope that our results help to identify new strategies for the development of antibacterial agents, which could complement conventional antibiotics by limiting cellular damage induced by bacterial enzymes", explains Dikic the high medical relevance of their discovery. The group of Ivan Dikic is located at both the Institute of Biochemistry II and the Buchmann Institute for Molecular Life Sciences at Goethe University Frankfurt and has previously contributed significantly to a paradigm change in the ubiquitin field. Ivan hypothesized early on that ubiquitin signals are recognized and translated by specialized domains in other proteins. He identified ubiquitin-binding domains in more than 200 ubiquitin receptors and was able to prove their role in diseases like cancer, amyotrophic lateral sclerosis and Parkinson's.


Dikic I.,Institute of Biochemistry II | Bremm A.,Institute of Biochemistry II
EMBO Journal | Year: 2014

The Parkinson's disease-associated ubiquitin E3 ligase parkin impacts various cellular processes including the autophagic clearance of defective mitochondria. In this issue of The EMBO Journal, Durcan et al () reveal a novel control mechanism of parkin-mediated mitophagy via the selective removal of atypical K6-linked ubiquitin chains from parkin by the deubiquitinase USP8. Together with recent studies on USP15 and USP30, this establishes a functional role for deubiquitination in mitophagy regulation. © 2014 The Authors.


Husnjak K.,Institute of Biochemistry II | Dikic I.,Institute of Biochemistry II | Dikic I.,Goethe University Frankfurt | Dikic I.,University of Split
Annual Review of Biochemistry | Year: 2012

Ubiquitin acts as a versatile cellular signal that controls a wide range of biological processes including protein degradation, DNA repair, endocytosis, autophagy, transcription, immunity, and inflammation. The specificity of ubiquitin signaling is achieved by alternative conjugation signals (monoubiquitin and ubiquitin chains) and interactions with ubiquitin-binding proteins (known as ubiquitin receptors) that decode ubiquitinated target signals into biochemical cascades in the cell. Herein, we review the current knowledge pertaining to the structural and functional features of ubiquitin-binding proteins and the mechanisms by which they recognize various types of ubiquitin topologies. The combinatorial use of diverse ubiquitin-binding domains (UBDs) in full-length proteins, selective recognition of chains with distinct linkages and length, and posttranslational modifications of ubiquitin receptors or multivalent interactions within protein complexes illustrate a few mechanisms by which a circuitry of signaling networks can be rewired by ubiquitin-binding proteins to control cellular functions in vivo. © 2012 by Annual Reviews. All rights reserved.


Razawi H.,Institute of Biochemistry II | Kinlough C.L.,Renal Electrolyte Division | Kinlough C.L.,University of Pittsburgh | Staubach S.,Institute of Biochemistry II | And 8 more authors.
Glycobiology | Year: 2013

The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111-1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [ 35S]Met/Cys or glycans with [3H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [3H]MUC1 when compared with [ 35S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1). © 2013 The Author 2013.


Hanisch F.-G.,Institute of Biochemistry II | Hanisch F.-G.,University of Cologne
Analytical Chemistry | Year: 2011

The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17-18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154. © 2011 American Chemical Society.


The attachment of ubiquitin was long considered as giving the "kiss of death", labelling superfluous proteins for disposal within a cell. However, by now it has been well established that ubiquitin fulfils numerous additional duties in cellular signal transduction. A team of scientists under the lead of Ivan Dikic, Director of the Institute of Biochemistry II at Goethe University Frankfurt, has now discovered a novel mechanism of ubiquitination, by which Legionella bacteria can seize control over their host cells. Legionella causes deadly pneumonia in immunocompromised patients. According to the current understanding, the coordinated action of three enzymes is needed for attaching ubiquitin to other proteins. In April this year, U.S. scientists described an ubiquitination reaction that depends only on a single enzyme from Legionella bacteria. The Dikic team together with the group of Ivan Matic (Max Planck Institute for Biology of Ageing, Cologne, Germany) now elucidated the underlying molecular mechanism of its action and revealed a hitherto unknown type of chemical linkage between ubiquitin and target proteins. Their discovery breaks new ground in the field. Sagar Bhogaraju, researcher in the Dikic laboratory, comments: "Most exciting is of course the question if this unusual ubiquitination also occurs in human cells independently of bacterial infection and if there are similar, so far unknown enzymes in humans, which may have a profound influence on cellular processes." When studying the new mechanism in more detail, the Frankfurt scientists were very surprised to find that the Legionella enzyme does not only transfer ubiquitin onto target proteins, but also chemically manipulates the remaining pool of ubiquitin molecules. Modified ubiquitin almost completely inhibits the conventional ubiquitination system, thereby revealing a new role for this enzyme during Legionella infections. Several important cellular processes are affected by this shut-down of the ubiquitination system, which can also cause a rapid cell death. The Dikic team showed for example that modified ubiquitin prevents degradation of mitochondria (a process called mitophagy), affects transduction of inflammatory signals and constrains protein degradation. "Most likely, Legionella is not the only bacterium using this mechanism. We hope that our results help to identify new strategies for the development of antibacterial agents, which could complement conventional antibiotics by limiting cellular damage induced by bacterial enzymes", explains Dikic the high medical relevance of their discovery. The group of Ivan Dikic is located at both the Institute of Biochemistry II and the Buchmann Institute for Molecular Life Sciences at Goethe University Frankfurt and has previously contributed significantly to a paradigm change in the ubiquitin field. Ivan hypothesized early on that ubiquitin signals are recognized and translated by specialized domains in other proteins. He identified ubiquitin-binding domains in more than 200 ubiquitin receptors and was able to prove their role in diseases like cancer, amyotrophic lateral sclerosis and Parkinson's.

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