Forschungsgruppe Herrlich

(Zur Zeit z.T. nur in Englisch verfügbar)

Krebszellbiologie

The Herrlich Laboratory: the control of cellular migration and growth

How do embryonic cells start migrating, what governs migration of stem cells during regeneration, how do tumor cells metastasize. This is at the core of the lab´s interest. Currently two genes are in the research focus: CD44 and Trip6.

CD44: the gene for CD44 is multiply alternatively spliced, therefore encodes a large family of type I transmembrane proteins. The splice variation addresses the extracellular portion. One of the splice variants, CD44v6 serves as an essential co-receptor for a tyrosine kinase which regulates migration: Met. In addition, CD44 mediates contact inhibition, the signal which makes cells stop growing, and CD44 may act also as tumor suppressor. This seemingly contradictory panel of actions need to be dissected. CD44 is also cleaved by g-secretase, like the Alzheimer APP protein. The cytoplasmic tail is taken up into the nucleus and activates a gene expression program involved in migration. We wish to find out, what regulates the cleavage and what exactly is the function in the nucleus. The methods include cellular and mouse models, structural biology and the identification of interference strategies, e.g. in order to prevent cleavage or to inhibit metastasis formation.

Trip6: Trip6 was discovered as a protein interacting with nuclear receptors. The nuclear form is generated by alternative translational start which eliminates the nuclear export sequence. The long form is located at focal adhesions at the plasma membrane. Suppression of the nuclear form reduces the efficiency of transcription driven by the Fos:Jun or NF-kB transcription factors and inhibits the antiinflammatory action of the glucocorticoid receptor. Nuclear Trip6 appears to serve as a platform on promoters by interaction with Fos, p65 (NF-kB) and nuclear receptors. The puzzle is, how it confers activating and repressing function. Both the nuclear and the cytoplasmic Trip6 isoforms appear to be engaged in growth control and migration.

Viral oncogenesis

Adenoviruses can transform rodent cells, catalyzed by the nuclear viral oncogenes E1A and E1B. We discovered a new action of E1A. It activates the Jun N-terminal kinase pathway and thus induces the transcription of Jun and ATF2 dependent genes. It appears that a small fraction of E1A is not in the nucleus, but rather at the plasma membrane where it causes the GTP loading and thus activation of the small G-proteins Rac and Cdc42. We wish to find out, how E1A does this. The methodology involves protein interaction screens and biochemical analysis, e.g. assays for guanine exchange factors and GTPase activating proteins.

Redox regulation of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) just like tyrosine kinases regulate numerous processes in cells. Their active enzyme centre carries a redox-sensitive cysteine and it is believed that inactivation by oxidation and activation by reduction is physiologically used for regulation. We found, however, that oxidative treatments by irradiating cells induces a calpain-dependent cleavage of PTPs. The current goal is to explore the mechanism and role of this cleavage reaction.

Inhouse collaborations with Aspasia Ploubidou, Zhao-Qi Wang, Helen Morrison, Jan Tuckermann, external collaboration with Frank Boehmer, Jena, and Olivier Kassel, Karlsruhe.

Ausgewählte Publikationen

Morrison H, Sperka T, Manent J, Giovannini M, Ponta H, Herrlich P (2007) Merlin/neurofibromatosis type 2 suppresses growth by inhibiting the activation of Ras and Rac. Cancer Res. 67, 520-527. [PubMed]
Orian-Rousseau V, Morrison H, Matzke A, Kastilan T, Pace G, Herrlich P, Ponta H (2007) Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol Biol Cell. 18, 76-83. [PubMed]
Jin H, Sperka T, Herrlich P, Morrison H (2006) Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase. Nature. 442, 576-579. [PubMed]
Matzke A, Herrlich P, Ponta H, Orian-Rousseau V (2005) A five-amino-acid peptide blocks Met- and Ron-dependent cell migration. Cancer Res. 65, 6105-6110. [PubMed]
Pust S, Morrison H, Wehland J, Sechi AS, Herrlich P (2005) Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell. EMBO J. 24, 1287-1300. [PubMed]
Fieber C, Baumann P, Vallon R, Termeer C, Simon JC, Hofmann M, Angel P, Herrlich P, Sleeman JP (2004) Hyaluronan-oligosaccharide-induced transcription of metalloproteases. J Cell Sci. 117, 359-367. [PubMed]
Kassel O, Schneider S, Heilbock C, Litfin M, Göttlicher M, Herrlich P (2004) A nuclear isoform of the focal adhesion LIM-domain protein Trip6 integrates activating and repressing signals at AP-1- and NF-kappaB-regulated promoters. Genes Dev. 18, 2518-2528. [PubMed]
Herrlich P, Morrison H, Sleeman J, Orian-Rousseau V, König H, Weg-Remers S, Ponta H (2000) CD44 acts both as a growth- and invasiveness-promoting molecule and as a tumor-suppressing cofactor. Ann NY Acad Sci. 910, 106-118. [PubMed]

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Forschungsgruppe Schilling: Neurodegeneration: Die Rolle der Proteolyse und abgeleitete Therapieansätze für die Huntington'sche Krankheit

Proteolytic processing of polyglutamine proteins

» WAYS to the involved protease and the cleavage site: Methods we are working on to narrow down the protease involved in cleavage of N-terminal Huntingtin and identify the cleavage site. Here we focus on protease inhibitors.

The importance of huntingtin (htt) cleavage has been demonstrated in many in vitro cell culture studies, showing that short fragments of htt more toxic than long fragments. Several in vivo studies confirmed the idea that HD mouse models show similar phenomena, in which full length HD mouse models did not show obvious toxicity while models with shorter fragments of htt did.

To examine the biology of mutant huntingtin (htt) in vivo we generated transgenic mice of the first 171, amino acids of htt including 18Q (normal repeat), 44Q (expanded repeat) and 82Q (causes juvenile HD). In transgenic mice expressing the N171-82Q protein, we observed motor impairment, loss of weight, hypoactivity and premature death. The neuronal loss is limited and not very prevalent but there is an accumulation of huntingtin reactivity in multiple populations of neurons and discrete nucelear and cytoplasmic aggregates, which are much more frequent. These behavioral abnormalities and neuropathological features were not observed in the either the N171-18Q or N171-44Q mice.

Western blot analysis of this N171 transgenic model revealed a N-terminal truncation site which cleaved about ~10 kDa in vitro and in HD brains, we decided to develop an array of antibodies to investigate the size.

Preliminary studies in HD postmortem brains revealed a similar sized proteolytic fragment, which aggregates in the nucleus and in HD mouse brains section. We conclude that there may be an proteolytic event of huntingtin in our HD mouse model and HD postmortem brain.

Proteolytic processing has been shown to be important in a DRPLA mouse model which we generated and observed truncation of the normal and expanded repeats. These full lengths atrophin-1(the DRPLA protein product) mouse model with 26Q (normal) and 65Q (expanded) exhibited proteolytic processing of both transgenes right after the polyglutamine repeat, leading to an N-terminal aggregated truncation product in the nucleus of the neurons since it has lost the nuclear export signal and can not get transported out. Similar sized fragments were found in nuclear fractionations of DRPLA postmortem brains. We believe that in HD and DRPLA proteolytic processing may generate smaller sized proteins of huntingtin and atrophin-1 which include the polyglutamine tract and are more likelely to aggregate.

Projekte

Identification of the truncation site in huntingtin and atrophin-1
Develop HD transgenic mice which lack the proteolytic site
Identify the protease
Test protease inhibitors in cell culture

Ausgewählte Publikationen

Schilling G, Savonenko AV, Klevytska A, Morton JL, Tucker SM, Poirier M, Gale A, Chan N, Gonzales V, Slunt HH, Coonfield ML, Jenkins NA, Copeland NG, Ross CA, Borchelt DR (2004) Nuclear-targeting of mutant huntingtin fragments produces Huntington's disease-like phenotypes in transgenic mice. Hum. Mol. Genet. 13, 1599-1610. [PubMed]
Schilling G, Coonfield ML, Ross CA, Borchelt DR (2001) Coenzyme Q10 and remacemide hydrochloride ameliorate motor deficits in a Huntington's disease transgenic mouse model. Neurosci Lett. 315, 149-153. [PubMed]
Schilling G, Jinnah HA, Gonzales V, Coonfield ML, Kim Y, Wood JD, Price DL, Li XJ, Jenkins N, Copeland N, Moran T, Ross CA, Borchelt DR (2001) Distinct behavioral and neuropathological abnormalities in transgenic mouse models of HD and DRPLA. Neurobiol Dis. 8, 405-418. [PubMed]
Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet. 8, 397-407. [PubMed]
Schilling G, Wood JD, Duan K, Slunt HH, Gonzales V, Yamada M, Cooper JK, Margolis RL, Jenkins NA, Copeland NG, Takahashi H, Tsuji S, Price DL, Borchelt DR, Ross CA (1999) Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA. Neuron. 24, 275-286. [PubMed]

Last update: March 27, 2008

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Lab Eberhard Fritz: DNA-Reparatur und genomische Instabilität

Die Reparatur und - damit verknüpft - die korrekte Topologie der zellulären DNA sind wesentliche Parameter in der Stabilität des Genoms. Eine Instabilität des Genoms fördert oder bedingt zelluläre Alterung und altersbedingte Erkrankungen. Wir untersuchen derzeit 1.) Mechanismen der individuellen Strahlenempfindlichkeit und 2.) Funktionen von Topoisomerase III-Proteinen.

Recombination detection system

» Gamma-Strahlen-induzierte Apoptose in primären Blutzellen.

Mechnismen der individuellen Strahlenempfindlichkeit

In der Gesamtbevölkerung variiert die Empfindlichkeit gegenüber ionisierender Strahlung stark zwischen verschiedenen Individuen. Es wird geschätzt, daß bis zu 20% der Krebspatienten auf eine Strahlentherapie klinisch überempfindlich reagieren.
In einem Kooperationsprojekt mit klinischen Partnern untersuchen wir die zelluläre Strahlenempfindlichkeit isolierter Blutzellen von Krebspatienten. Verschiedene Endpunkte zellulärer Strahlenreaktionen (Comet assay, Apoptose, H2A-X-Phosphorlierung) ermöglichten die Charakterisierung besonders strahlen-resistenter oder -empfindlicher Blutzellen. Ob diese in vitro-Befunde mit der klinischen Strahlenempfindlichkeit der Patienten korrelieren, wird derzeit überprüft. Auf der Suche nach molekularen Ursachen der Strahlenempfindlichkeit untersuchen wir in diesen Zellen mögliche Kandidaten-Gene und -Proteine der DNA-Reparatur.

Funktionen von Topoisomerase III-Proteinen

Die Topoisomerasen III-alpha und -beta entwinden die DNA-Überstruktur und interagieren in Säugerzellen mit den RecQ-Helikasen WRN (Werner Syndrom-Helikase) und BLM (Bloom's Syndrom-Helikase). Vermutlich sind diese Proteinkomplexe für das Auflösen von Rekombinationsstrukturen verantwortlich; die genauen Funktionen dieser Topoisomerasen sind weitgehend unbekannt. Zur weiteren Funktionsaufklärung untersuchen wir 1.) das Auftreten katalytisch-aktiver Topoisomerase III-Poteine, die kovalent an DNA gebunden sind, sowie 2.) Zellen mit herunterregulierten Topoisomerase III-Proteinen.

Ausgewählte Publikationen

Greve B, Dreffke K, Rickinger A, Könemann S, Fritz E, Eckardt-Schupp F, Amler S, Sauerland C, Braselmann H, Sauter W, Illig T, Schmezer P, Gomolka M, Willich N, Bölling T (2009) Multicentric investigation of ionising radiation-induced cell death as a predictive parameter of individual radiosensitivity. Apoptosis. 14, 226-235. [PubMed]
Mierau M, Drexler GA, Kutzera A, Braunschmidt K, Ellwart J, Eckardt-Schupp F, Fritz E, Bachl J, Jungnickel B (2008) Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription. Nucleic Acids Res. epub ahead of print. [[PubMed]]
Gehrmann M, Marienhagen J, Eichholtz-Wirth H, Fritz E, Ellwart J, Jäätellä M, Zilch T, Multhoff G (2005) Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells. Cell Death Differ. 12, 38-51. [PubMed]
Philbrook C, Fritz E, Weiher H (2005) Expressional and functional studies of Wolframin, the gene function deficient in Wolfram syndrome, in mice and patient cells. Exp Gerontol. 40, 671-678. [PubMed]
Drexler GA, Rogge S, Eckardt-Schupp F, Zdzienicka M, Fritz E (2004) Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells. DNA Repair (Amst). 3, 1335-1343. [PubMed]
Drexler GA, Wilde S, Beisker W, Ellwart J, Eckardt-Schupp F, Fritz E (2004) The rate of extrachromosomal homologous recombination within a novel reporter plasmid depends on expression of the ATM protein. DNA Repair (Amst). 3, 1345-1353. [PubMed]
Eckardt-Schupp F, Mörtl S, Fritz E (2004) The network of radiation responses and genomic stability. In: Life Science and Radiation (Kiefer K, Ed), Springer Verlag Heidelberg, 57-68.
Fritz E, Digweed M (2002) Nijmegen breakage syndrome. In: Chromosomal Instability and Aging: Basic Science and Clinical Implication (Hisama F, Weisman S, Martin G, Hrsg), Verlag M Dekker, New York, 311-344.
Raderschall E, Bazarov A, Cao J, Lurz R, Smith A, Mann W, Ropers HH, Sedivy JM, Golub EI, Fritz E, Haaf T (2002) Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis. J Cell Sci. 115, 153-164. [PubMed]

Last update: 2. November 2009

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